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PUBLISHED FOR THE LIFE SCIENTIST BY LIFE TECHNOLOGIES, INC.
VOLUME 21 • NUMBER 3 • 1999
Transfection in 96–well Plates – 58 Mammalian Cell Lines for Transfection – 62 Fluorescent DNA Ladders – 64 Helpful Tips for Custom Primers – 69 Some Basics of Cell Culture Media – 76
c o n t e n t s
ABOUT THE COVER: Transfection in 96-well plates. (See page 58).
Jean-Pierre Pichet and Valentina Ciccarone Linda Roy, Sharon Cates, Kevin Schifferli, Jean-Pierre Pichet, Valentina Ciccarone, Shelley Bennett, and Pamela Hawley-Nelson
FOCUS contains manuscripts describing novel techniques, improvements of common techniques, simpliﬁed protocols, and troubleshooting.“Instructions to Authors” are available on the Internet or from the editor: FOCUS Editor: Dr. Doreen Cupo Life Technologies, Inc. 9800 Medical Center Drive Rockville, MD 20849-6482 (800) 828-6686 (301) 610-8000 outside the U.S. E-mail: firstname.lastname@example.org Assistant to the Editor: Karen Carstensen Salovich © Copyright Life Technologies®, Inc., 1999 FOCUS ® is published triannually by Life Technologies, Inc. POSTMASTER: Send address changes to FOCUS, Life Technologies, Inc. P.O. Box 6482, Rockville, MD 20849-6482 Requests for subscriptions and address changes should be directed to the nearest Life Technologies ofﬁce: U.S. ACADEMIC ORDERS To Order/TECH-LINE: SM (800) 828-6686 Fax: (800) 331-2286 U.S. GOVERNMENT ORDERS To Order: (888) 584-8929 Fax: (888) 584-8930 U.S. INDUSTRIAL ORDERS To Order/TECH-LINE: (800) 874-4226 Fax: (800) 352-1468 Internet www.lifetech.com email@example.com INTERNATIONAL ORDERS U.S.A. Ofﬁce for Latin America To Order/TECH-LINE: (301) 610-8709 Fax: (301) 610-8724 AUSTRALIA Melbourne To Order/TECH-LINE: 1800 331 627 Tel: (03) 9558 9622 Fax: (03) 9558 9722 CANADA Burlington, Ontario To Order: (800) 263-6236 TECH-LINE: (800) 757-8257 Fax: (800) 387-1007 EUROPE Paisley, Scotland To Order: 0800 269210 TECH-LINE: 0800 838380 Fax: 0800 243485 HONG KONG Tsuen Wan Tel: (852) 2407-8450 Fax: (852) 2408-2280/2409-6043 INDIA New Delhi To Order: 91-11-577-3282 Fax: 91-11-577-3281 JAPAN Tokyo Tel: (81-3) 3663-8241 Fax: (81-3) 3663-8242 NEW ZEALAND Auckland To Order/TECH-LINE: 0800 600 200 Tel: (09) 579 3024 Fax: (09) 579 3119 PEOPLE’S REPUBLIC OF CHINA Beijing To Order: (86-10) 6256-3836 Fax: (86-10) 6256-4852 PEOPLE’S REPUBLIC OF CHINA Shanghai To Order: (86-21) 6471-1313 Fax: (86-21) 6471-1313 TAIWAN Taipei Tel: (886-2) 2651-6156 Fax: (886-2) 2653-8100 Editorial Review Board: Holly Anderson, Paul Battista, Stephen Gorﬁen, James Hartley, Curtis Henrich, Larry Mertz, Frank Swartzwelder Contributing Editorial Reviewers: J.J. Lin, Dave Schuster
Transfection of Mammalian Cells in 96-Well Plates with LIPOFECTAMINE ™ 2000 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Cationic Lipid Reagent Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 High Transfection Efﬁciency of Cloned Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
DNA Ladders for Fluorescent Fragment Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Heather Jordan and Joseph Solus
One-Step RT-PCR to Detect Cytokine/Chemokine Induction in Macrophages . . . . . . . . . . . . . . . . . 66
Monique Bongers, Ekke Liehl, and Johannes Barsig
Helpful Tips for Custom Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
AFLP ™ Analysis of the Fruit Fly Ceratitis capitata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Gino Corsini, Augusto Manubens, Manuel Lladser, Sergio Lobos, Daniela Seelenfreund, and Carlos Lobos
Buffer Compatibility for Common Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Joe Crouse, Teresa Myers, and Julie Brent
How Basal Media Provide an Optimal Growth Environment for Cell Culture . . . . . . . . . . . . . . . . 76
AFLP™ is a trademark of Keygene n.v. CFLP™ is a trademark of Third Wave Technologies, Inc. GeneScan® is a registered trademark of The PerkinElmer Corporation, Inc. Kodak® is a registered trademark of Eastman Kodak Co. Triton® is a registered trademark of Rohm & Haas, Co. TRIZOL® is a registered trademark of Molecular Research Center, Inc. Tween 20® is a registered trademark of ICI Americas, Inc. Purchase of Taq DNA Polymerase is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research and development in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. This product is sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., and The Perkin-Elmer Corporation Inc.
99-142 Part No. 53077
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Essential Technologies for the Science of Life™
F O C U S (1 9 9 9)
V O L U M E
N U M B E R 3
or a "One-day Protocol. No." where a cell suspension is added to the DNA-reagent complexes prepared in the wells.6 320 240 160 DNA 80 (ng) 160 – 80 – FIGURE 1. 1.2 0.2 0. duplicate plates were assayed with X-gal (panel A) and ONPG (panel B). The sensitivity of these protocols to measure gene activity in cDNA libraries is also shown.0 1. with 584 mg/L Lglutamine and 15 mg/L phenol red) supplemented with 0. For COS and 293 cells. Cells were transfected with pCMV•SPORT-βgal DNA and LIPOFECTAMINE 2000 Reagent.8 1. Inc.8 1. COS-7L (Cat. 293-H.4 0. sera.8 1.5 × 104.8 1. 4 × 104 to 6 × 104 cells/well).6 0. No.000 1." where cells are plated the day before transfection.6 0. Cell cultures were maintained at 37°C in a humidiﬁed.6 0. Maryland 20849 Transfection of Mammalian Cells in 96-Well Plates with LIPOFECTAMINE ™ 2000 Reagent L IPOFECTAMINE 2000 Reagent Panel A 293-H Cells 320 – 18. 58 80 – 320 240 160 DNA 80 (ng) LIPOFECTAMINE 2000 Reagent (µl) CHO-S Cells 320 – 4. this new reagent is especially suited for transfections in the 96-well format. In this report. At 24 h after complex addition.000 4.8 1.6 0. Results from the optimal seeding density for each cell line are shown: 2 × 104 for CHO-S.5 × 104 to 2. TRANSFECTION.500 3. resulting in high levels of recombinant protein expression (1).2 0.2 METHODS CELL CULTURE.000 2.500 1.8 1.2 320 240 160 DNA 80 (ng) LIPOFECTAMINE 2000 Reagent (µl) COS-7L Cells 320 – 3. 5% CO2 incubator. and reagents were from Life Technologies.0 1. poly-D-Lysine coated plates were used for transfection protocols. All cell lines. Rockville.000 12.4 0.0 1.500 2. and 5 × 104 for 293-H.1 mM nonessential amino acids (NEAA) and 10% FBS.000 0 0. The protocol for transfection of cells with LIPOFECTAMINE 2000 Reagent involves very few steps and can be performed in serum-containing medium.4 0. CHO-S (Cat.000 DNA (ng) 240 – ng β-gal/cm2 14. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . For the Standard Protocol.500 240 – 2. we optimized transfection conditions for 96-well plates with the 3 cell lines commonly used in high-throughput protocols—CHO.000 0. which is ideal for high-throughput protocols.4 0. 11631) cells (2) were cultured in Dulbecco’s MEM (D-MEM High Glucose: 4. 2 × 104 to 4 × 104. 80 – LIPOFECTAMINE 2000 Reagent (µl) 0.000 DNA (ng) 240 – ng β-gal/cm2 3.4 160 – Panel B DNA (ng) 160 – ng β-gal/cm2 transfects cells at very high efﬁciencies. COS-7L.000 16.500 4. Therefore.2 0.0 1.0 1.000 2.2 2. 11622). Transfection of cells using the standard protocol.2 0 LIPOFECTAMINE 2000 Reagent (µl) 0.2 0 LIPOFECTAMINE 2000 Reagent (µl) 0. culture media.000 6.000 500 0. Several seeding densities were tested for each cell type (CHO-S.2 0.000 8.t r a n s f e c t i o n Jean-Pierre Pichet Valentina Ciccarone Molecular Biology Research and Development Life Technologies.500 mg/L D-glucose.000 1.000 500 0. No.0 1. 11619).4 0. 2. and 293-H (Cat.500 1.2 0.6 0. COS.5 × 104 for COS-7L. and 293—using either a "Standard Protocol.2 LIPOFECTAMINE 2000 Reagent (µl) 0.000 10. cells were plated in 96-well plates the day before transfection in 100 µl of growth medium.
before mixing with the diluted DNA. DNA and LIPOFECTAMINE 2000 Reagent (Cat. Optimal transfection conditions of cells in 96-well plates using the standard protocol.8 0. At optimal conditions. DNA-LIPOFECTAMINE 2000 Reagent complexes were prepared in 96-well plates and cell suspension added to each well. The optimal cell concentration for the rapid.0 0.000 2. table 1).2 × 105 TABLE 2. the expression levels were good. ONE-DAY PROTOCOL.2 0.1% Triton® X 100 to measure β-gal enzymatic activity using ONPG (4). No.5 × 104 5 × 104 DNA (ng/well) 240 320 320 LIPOFECTAMINE 2000 Reagent (µl/well) 1 1 1 TABLE 1. 19586) and pCMV•SPORT6-βgal-neo plasmid DNA were puriﬁed using the CONCERT™ High Purity Plasmid Puriﬁcation Maxiprep System (Cat. 11452).2 0. One-day Protocol where the complexes are prepared in a 96-well plate and a cell suspension is added directly to the complexes was evaluated.000 8.6–0. and a 100-µl cell suspension was added to the complexes in wells. in 96-well plates. For each well.6 0. it is not necessary to plate the cells the day before transfection. To determine optimal conditions for transfection in a 96-well plate. one-day protocol.500 1.4 ng β-gal/cm2 3. LIPOFECTAMINE 2000 Reagent and DNA F O C U S (1 9 9 9) V O L U M E 2 1 Cell Line CHO-S COS-7L 293-H Seeding Density (cells/well) 2 × 104 2.8 0.8 2. concentration were evaluated at 3 cell densities.4 500 320 240 160 DNA 80 (ng) 0 0.0). At 24 h post-transfection. The diluted LIPOFECTAMINE 2000 Reagent was incubated for 5 min at room temperature. pCMV•SPORT6βgal-neo was serially diluted into total plasmid DNA from a human brain cDNA library.000 16.000 12.t r a n s f e c t i o n pCMV•SPORT-βgal (Cat. 11668) were ﬁrst diluted separately in 25 µl OPTI-MEM® I Reduced Serum Medium without serum. No.000 1. RESULTS AND DISCUSSION STANDARD PROTOCOL.000 6. were incubated at room temperature for 20 min for complex formation. so this rapid protocol may not be as useful with this cell line. Good transfection efﬁciencies were obtained with the 3 cell lines. and time savings may provide an advantage in some applications. The maximum activity for 293 and COS-7 cells was lower than with the Standard Protocol (∼40% for 293 and ∼50% for COS-7). No.000 14. In this protocol. Optimal transfection conditions of cells with the rapid. A more rapid. Cells were trypsinized.000 COS-7L Cells 0. For CHO cells. 0. One-day Protocol was ∼2. CDNA LIBRARY SCREEN.000 ng β-gal/cm2 10. DNA-reagent complexes were prepared as described above in 96-well plates. and optimal ranges were identiﬁed (ﬁgure 1. 50 µl of complex was added directly to the cells in their growth medium and gently mixed. The transfection was performed with the Standard Protocol.5 times higher than for the Standard Protocol (ﬁgure 2.000 0 0. the cell concentrations were 1. One-day Protocol.000 4. Transfection of cells using the rapid.0 1. Cells were frozen in lysis buffer at −80°C for at least 1 h prior to assay. CDNA LIBRARY SCREENING.2 320 240 160 DNA 80 (ng) LIPOFECTAMINE 2000 Reagent (µl) LIPOFECTAMINE 2000 Reagent (µl) FIGURE 2.6 1. Using duplicate plates for each cell line. Ranges of DNA and reagent concentrations at 3 cell densities were evaluated to determine optimal conditions. The amount of pCMV•SPORT6-βgal-neo DNA in the wells varied from 156 pg to 320 ng. β-GAL ACTIVITY. The DNA-reagent complexes.2 × 105 cells/well for 293-H cells and 6 × 104 cells/well for COS-7L cells in a total volume of 100 µl of growth medium. the cells were rinsed with D-PBS and either ﬁxed and stained in situ with X-gal (3) or lysed and harvested in 150 µl of 0. table 2). the activity was ∼70% lower.000 2. The standard protocol was used to determine the sensitivity 59 N U M B E R 3 .4 1. Cell Line CHO-S COS-7L 293-H Seeding Density (cells/well) 5 × 104 6 × 10 4 DNA (ng/well) 320 320 320 LIPOFECTAMINE 2000 Reagent (µl/well) 0.8 2. 293-H Cells 18. However. The total DNA amount transfected was constant in all wells at 320 ng.2 0. For the rapid.1 M Tris-HCl (pH 8.500 2. one-day protocol.
L.25 0. New York. Hawley-Nelson. L. and Hawley-Nelson. Schifferli.F. K.313 0. (1989) Molecular Cloning: A Laboratory Manual.000 8.000 0 320 160 80 40 20 10 5 2. 60 In summary.25 0. J..66.. J. S. J. V. Second Edition. p.F.. For example. (1986) EMBO J. Duplicate plates were assayed with X-gal (panel A) and ONPG (panel B). Pichet. and Nicolas. Wells in each column of the plate contained the same amount of pCMV•SPORT6-βgal-neo DNA. 4. 62. Ray Hadley and Dr.5 1. 5.000 ng β-gal/cm2 6. 54. and Maniatis.. of detection of gene expression in a cDNA library. 2. V.000 ng β-gal/cm2 3. P. Ciccarone.625 0.e.000 2.5 1.156 320 160 80 40 20 10 5 2... The total amount of DNA transfected was always 320 ng. and Bennett. Fritsch. the better COS-7L sensitivity was due to the SV40 origin of replication in the plasmid allowing higher expression levels in the SV40-transformed COS-7L cells. suggesting that this protocol may be used for screening to detect speciﬁc gene expression in cDNA libraries. Evans..-P. J. S. 3133.P.000 Panel B 5. Cates.000 10. luminescence) may allow greater sensitivity.. Starting with the protocols described here. Cells plated at their optimal seeding density were transfected with serially diluted pCMV•SPORT6-βgal-neo added to plasmid DNA from a cDNA library. FOCUS REFERENCES 1. Chris Gruber for the brain library cDNA..625 0.. L. Chu. Sanes. 3.25 0. Bennett.R. K. Roy. Schifferli. Roy.5 1.000 0 320 160 80 40 20 10 5 2.000 4.. Ciccarone. Cold Spring Harbor Press..000 2...313 0.156 Target DNA (ng) Target DNA (ng) 293-H Cells 12. Other detection methods (i. P. T. Y. (1999) FOCUS 21.156 Target DNA (ng) Target DNA (ng) FIGURE 3. Sambrook. J.000 1. transfection conditions can be easily established for automated or robotic systems in highthroughput screening applications. Expression was detected with small amounts of β-gal plasmid (ﬁgure 3). E.25 0. K.625 0.000 4. Rubenstein..313 0..t r a n s f e c t i o n Transfection of Mammalian Cells continued Panel A COS-7L Cells 6. S.. Detection levels will depend on the vector used and the assay sensitivity.625 0. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . (1999) FOCUS 21. Detection of gene expression in a cDNA library. LIPOFECTAMINE 2000 Reagent is highly efﬁcient for transfection of cells in a 96-well format.156 320 160 80 40 20 10 5 2. ACKNOWLEDGEMENTS The authors thank Dr. 16. Data in panel B are the mean ± SD for N = 8.131 0.5 1. Cold Spring Harbor.R. Pichet.
Application Difﬁcult-to-transfect cells Most adherent cells Adherent cells in the presence of serum Suspension cells Insect cells Endothelial cells RNA Oligonucleotides Recommended Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent or LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent DMRIE-C Reagent CELLFECTIN® Reagent LIPOFECTIN® Reagent DMRIE-C Reagent LIPOFECTIN Reagent.com and available from the TECH-LINE. the general guidelines to follow in selecting a reagent are summarized at the right.* Please consult our web site (www.lifetech. LIPOFECTAMINE Reagent. The table below contains recommended cationic lipid reagents for high-efﬁciency transfection of key cell lines.t r a n s f e c t i o n Cationic Lipid Reagent Selection W Cell Line 293-F 293-H BE(2)C BHK-21 CHO-K1 hen starting transfections in your lab. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 61 . or CELLFECTIN Reagent Mammalian Cell Type Human kidney Human kidney Human neuroblastoma Hamster kidney Hamster ovary Hamster ovary Hamster ovary Monkey kidney Monkey kidney Human primary passaged Human cervical cancer Human colon cancer Human ﬁbrosarcoma Human endothelial Human endothelial Human primary passaged Dog kidney Human lung Mouse ﬁbroblasts Rat pheochromocytoma Human breast cancer Monkey kidney Insect Cell Type Drosophila melanogaster Spodoptera frugiperda Spodoptera frugiperda Recommended Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent DMRIE-C Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTIN with PLUS Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent (with serum) LIPOFECTIN Reagent LIPOFECTIN Reagent LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (without serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE PLUS Reagent LIPOFECTAMINE 2000 Reagent LIPOFECTAMINE 2000 Reagent (with serum) LIPOFECTAMINE PLUS Reagent (without serum) LIPOFECTAMINE 2000 Reagent (without serum) Recommended Reagent CELLFECTIN Reagent CELLFECTIN Reagent CELLFECTIN Reagent CHO-S (adherent) CHO-S (suspension) (In CD-CHO Medium) COS-1 COS-7L Fibroblasts HeLa HT-29 HT-1080 HUAEC (primary) HUVEC (primary) Keratinocytes (In Keratinocyte-SFM) MDCK MRC-5 NIH-3T3 PC-12 SK-BR3 Vero Cell Line D.lifetech.com) for the most current additions to our recommended reagent table.Mel-2 Sf9 Sf21 * Protocols for these transfections are in the TECH-ONLINE section of our web site at www.
A and reagents were from Life Technologies unless otherwise noted. The parental lines were obtained as follows: 293-H (from Leaf Huang at the University of Pittsburgh). CHO-S (Cat. Sharon Cates. The transfections with LIPOFECTAMINE 2000 Reagent (5) were performed in growth medium (D-MEM/NEAA/10% FBS). For transfection. Alternatively. cultured. TRANSFECTION. After 5 min. C METHODS CELL CULTURE.1 M Tris-HCl (pH 8. the DNA was precomplexed with the PLUS Reagent by diluting 0. b-GAL ACTIVITY. with 584 mg/L L-glutamine and 15 mg/L phenol red) supplemented with 0. we show that these cell lines exhibit higher transfection activity than other available cells. The DNA/PLUS Reagent was mixed with the diluted LIPOFECTAMINE Reagent and incubated for 15 min at room temperature. The precomplexes were incubated at room temperature for 15 min. the diluted DNA was added to the diluted LIPOFECTAMINE 2000 Reagent and incubated for 15 min at room temperature.1 mM non-essential amino acids (NEAA) and 10% FBS at 37°C in 5% CO2. The DNA was prepared using the CONCERT™ High Purity Maxiprep System. As a result. 11631). At 24 h after addition of complexes. cells were rinsed once with Dulbecco’s Phosphate Buffered Saline Solution and lysed in 0. Stained cells were photographed using a 10X objective on a Nikon inverted microscope with Hoffman optics. Since these immortalized cell lines are aneuploid.8 µg DNA to 17 µl with D-MEM/ NEAA and adding 8 µl PLUS Reagent. COS-7 (2) cells. No. For transfection with LIPOFECTAMINE PLUS Reagent (4). 11619). CHO-S (from Robert Tobey at Los Alamos).0 × 105 HEK 293 cells. Jean-Pierre Pichet. 0 to 6 µl of LIPOFECTAMINE 2000 Reagent were diluted to 50 µl with DMEM/NEAA. Life Technologies has used several better-transfecting sublines to isolate clones exhibiting high transfection efﬁciency. In this paper. variability in transfection efﬁciency has been observed. After many passages. and COS-7L (Cat. Cell extracts were thawed and assayed for β-gal activity using ONPG (7). 0 to 5 µl of LIPOFECTAMINE Reagent were diluted to 25 µl with D-MEM/NEAA and incubated for 5 min at room temperature. Shelley Bennett. Inc. While these cell lines have been available for many years. The day before the transfection. and banked at American Type Culture Collection (ATCC) or other cell banks. The complexes (100 µl) were added to each well. Expression of β-gal. No. 62 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . 500 µl of D-MEM/NEAA containing 20% FBS were added to the cells. sublines have evolved in various laboratories that are subtly different from the cells originally isolated. No. and 8. The growth medium was removed from the cells and replaced with 500 µl of D-MEM/NEAA. Maryland 20849 Transfection High Cell Lines Efﬁciency of Cloned hinese Hamster Ovary (CHO) (1) cells. high glucose: 4. ATCC cells were used <10 passages after thawing. 11668) or LIPOFECTAMINE PLUS™ Reagent according to manufacturer’s recommendations. All cell culture media. 11622). cells were ﬁxed and stained in situ with X-gal (8). Protein was determined by a Bradford assay. The cells photographed are the peak activity from a dose response. Kevin Schifferli. Life Technologies cell lines used were 293-H (Cat. they were plated on poly-D-lysine (50 µg/well) coated plates. 0. and the cells were placed at 37°C in 5% CO2. B FIGURE 1. Percent stained cells was determined by counting 3 ﬁelds and averaging. 293 cells from Life Technologies (panel A) or ATCC (panel B) were transfected using LIPOFECTAMINE 2000 Reagent and assayed after 24 h.t r a n s f e c t i o n Linda Roy. and Pamela Hawley-Nelson Research and Development Life Technologies. the transfection efﬁciency may change (9).500 mg/L D-glucose. 2. The complexes were added to the cells and incubated for 4 h in 37°C. No. expression. and COS-7L (from Tom Livelli at Specialty Media).5 × 105 CHO cells. sera.8 µg) was diluted to 50 µl with D-MEM/NEAA. Rockville. Valentina Ciccarone. and HEK 293 (3) cells are among the most commonly used mammalian cell lines for transfection. and large-scale production of recombinant proteins. No. the commercial lines have not always been easily transfected.1% Triton® X-100 (v/v) and frozen at −70°C overnight. 11625). Some researchers have selected for more easily transfected variants. We recommend Life Technologies cells for up to 30 passages or 3 months post-thaw.0 × 104 COS-7 cells were plated in 0. Since 293 and COS-7 cells are weakly adherent cells. 293-F (Cat. The cells were transfected with pCMV•SPORT-βgal DNA using LIPOFECTAMINE™ 2000 Reagent (Cat.0). 293-F (from Robert Horlick at Pharmacopoeia). Meanwhile. cells were grown as adherent cultures in Dulbecco’s MEM (D-MEM. 1. 5% CO2. DNA (0.5 ml of growth medium in each well of 24-well plates.
Do not freeze the reagents.-J. How does LIPOFECTAMINE 2000 Reagent improve transfection compared to other reagents? A. Q. Chu. 16. Cold Spring Harbor. (1986) EMBO J. Glutzman. K. Graham. P. Virol. Higher levels of protein expression have been observed in most of the cell lines tested. these cells are provided adapted to serumfree medium and suspension culture. Masoud.F. New York.R. Comparison of β-gal activity. 175. 5. P.-P. L. TABLE 1. No. J. Ciccarone. K. V. 3. (1999) Focus 21. Schifferli. Schifferli. Roy.. Sanes.. Q. V. 9. the streamlined protocol allows the addition of complexes directly to cells without changing media.. 293-F. V. T. Evans.L. For CHO-S cells. (1999) Focus 21.. the CHO-S. 8. (1997) Focus 19. Cells were stained for β-gal at 24 h after addition of complexes.. 7. Similarly. While penicillin and streptomycin are not toxic to eukaryotic cells. Pichet. 54. it also allows transient transfection in suspension culture and the chemically deﬁned CDCHO Medium (6).. 5. the increased cell permeability occurring during transfection leads to much higher levels of antibiotics getting into cells. (1977) J. Optimize the amount of each reagent. J. Second Edition. K.. The magnitude of difference from cells not selected for transfection ability was affected by the transfection reagent. P. 293-H. Lan. L. and Shih. Gen. (1958) J. In addition.. and Maniatis. Why do you recommend using medium without antibiotics during transfection? A. because proteins can interfere with complex formation. P. Y. with LIPOFECTAMINE 2000 Reagent demonstrating >95% transfected cells for the Life Technologies cells.. Q. F.. p. 52. 160 140 120 ng β-Gal/µg protein ng β-Gal/µg protein 100 80 60 40 20 0 293-F 293-H HEK 293 FIGURE 2. Is it necessary to use medium without serum during transfection? A. 3133.. Cold Spring Harbor Press. Results indicated that the clones selected for transfectability had high peak transfection efﬁciency (ﬁgure 1.. Transfection efﬁciency. E.. This facilitates use of these cells for large-scale protein production after selection of a stably transfected clone. What is the shelf life of cationic lipid reagents? A. Lichaa. J.. It is essential to form the DNA/cationic lipid reagent complex in the absence of serum. 6. Shih. Hawley-Nelson. No. In contrast to the percent of F O C U S (1 9 9 9) V O L U M E 2 1 cells transfected. Peak transfection activity of Life Technologies (■) or ATCC (□) cell lines was determined with ONPG 24 h after addition of DNA/LIPOFECTAMINE 2000 Reagent complexes. 108.R. (1989) Molecular Cloning: A Laboratory Manual. F. Can I use the same amount of any cationic lipid reagent for my cell line? A. Q. (1981) Cell 23. 945. and Bennett.. K. M. This can lead to higher cell death. the CHO-S clone demonstrated almost twice the β-gal activity of the CHO-K1 cell line. and COS-7L cells were transfected better than ATCC cell lines..t r a n s f e c t i o n Cell Line COS-7 COS-7L 293 293-F 293-H CHO-K1 CHO-S Source ATCC Life Technologies ATCC Life Technologies Life Technologies ATCC Life Technologies Transfection Efﬁciency (% stained cells) LIPOFECTAMINE PLUS LIPOFECTAMINE 2000 Reagent Reagent 35 ± 2 74 ± 6 39 ± 9 99 ± 0 99 ± 0 45 ± 3 97 ± 2 73 ± 11 99 ± 0 73 ± 8 99 ± 0 99 ± 0 86 ± 2 96 ± 3 REFERENCES 1.. table 1). 16. This high efficiency was possible with transfection in the presence of serum with LIPOFECTAMINE 2000 Reagent. Exp. S. J. they can be added to cells in serumcontaining medium.. Hawley-Nelson..66. Stored at 4°C in a closed container. 293 ATCC 45 40 35 30 25 20 15 10 5 0 CHO COS The Help Box from Your Technical Support & Training Team Q. 36. FOCUS N U M B E R 3 63 . In summary. Puck. RESULTS AND DISCUSSION Cell lines were evaluated for transfection efﬁciency using 2 reagents. and Nicholas.-J. Evans K.F. T. and Hawley-Nelson. 319. Schifferli. Fritsch. J. Rubenstein. J. (1995) Focus 17. Jessee. 4. the peak β-gal activity was higher in the clones selected for transfection (ﬁgure 2). they are stable for 12 months. Once the complexes are formed. P. Y. Sambrook.. 2. Med. For added ﬂexibility. Ciccarone. 60. and Ciccarone.
The mixtures were heat denatured at 90°C for 2 min.500 1.25FA. The GENOTYPE TAMRA 50-500 DNA Ladder was analyzed by electrophoresis on an ABI 377 gel. heat denatured at 95°C for 5 min. The GENOTYPE DNA Ladders. For the software to distinguish adjacent lanes from one another.000 3. 64 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 .1.9994 4. Maryland 20849 DNA Ladders for Fluorescent Fragment Analysis luorescence-based DNA electrophoresis systems are used often in genotyping applications to analyze polymorphic markers whose alleles differ by discrete size increments. cooled on ice. Linear correlation of the GENOTYPE TAMRA 50-500 DNA Ladder. the instruments and software usually require that the size standard be electrophoresed in the same lane or capillary with each sample. 10 mM EDTA). Inc. For analysis.5 µl of blue dextran solution (50 mg/ml blue dextran.5 µl were electrophoresed on a 36-cm. If all lanes of the gel were loaded at the same time. cooled on ice.500 4.000 Automated Sequencer and were used to determine the size of PCR products ampliﬁed from dinucleotide repeat microsatellite markers using primer sets from Genethon published sequences as previously described (1). No. it becomes necessary to load every other lane of the gel.000 – 70 50 100 150 200 250 300 Size (bp) 350 400 450 500 FIGURE 2. Data were analyzed using GeneScan® 3. Data were analyzed using GeneScan 3. No. Rockville.75 µl of deionized formamide. 11726) and the GENOTYPE TAMRA 60-500 DNA Ladder (Cat. To determine the sizes of the unknown fragments.000 2. The ﬂuorescent fragments were detected as they passed by the scanning laser during electrophoresis. – 90 – 210 – 200 – 190 – 310 – 300 – 290 – 350 – 330 For gel-based systems.5 µl of sample or water were mixed with 2. 0.1 software. 10773) polyacrylamide sequencing gel using a 36-lane sharkstooth comb. 0. and load the remaining lanes of the gel. electrophorese the samples brieﬂy.e l e c t r o p h o r e s i s Heather Jordan and Joseph Solus Research and Development Life Technologies. R2 = 0.25 µl of the DNA ladder was mixed with 12 µl of deionized formamide.000 1. sharkstooth combs are used for higher sample throughput and easier loading of the thin gels.25% (GEL-MIX® 4. For capillary electrophoresis. This paper describes ﬂuorescently labeled DNA ladders that can be used individually or as a dual-ladder conﬁguration for simpliﬁed loading of gels with sharkstooth combs. The "Data Point" (Y axis) is a measure of the number of scans across the gel by the laser prior to detection of the fragment and thus corresponds to the migration rate of the fragment. No. the DNA bands of the sizing ladder would appear as continuous lines across the entire gel and adjacent lanes would not be distinguishable. 4. the ladders were analyzed on a POP-6 matrix under denaturing conditions using an ABI 310 Capillary Genetic Analyzer.1 software.500 Data Point 3. F b– 500 – 490 – 480 – 460 – –b – 500 – 490 – 470 – 450 440 – – 430 420 – – 410 400 – – 390 380 – – 370 360 – 350 – 340 – 320 – 300 – 280 – – 270 260 – – 250 240 – – 230 220 – 200 – 180 – – 170 160 – – 150 140 – – 130 120 – – 110 100 – 90 – 80 – 60 – – 50 FIGURE 1. An alternative to time-consuming multiple gel loadings is to use DNA ladders with different band sizes in adjacent lanes.25 µl of DNA ladder. and 0. and the entire sample injected.1. and 1. Cat. 1. Alternating lanes of a 4. the GENOTYPE TAMRA 50-500 DNA Ladder (Cat. METHODS For gel electrophoresis.25% denaturing polyacrylamide gel were loaded with the GENOTYPE TAMRA 60-500 DNA Ladder and the GENOTYPE TAMRA 50-500 DNA Ladder. 11727) were analyzed on the ABI 377XL 5.500 2.
94 232. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 65 . Several dinucleotide repeat microsatellite markers ampliﬁed from human DNA as previously described (1) were sized with the G ENO TYPE DNA Ladders. Cat. but may not account for all alleles in the human population.90 272..13 245.12 273.35 247.. The migration of bands was linear with respect to fragment size (ﬁgure 2). FOCUS REFERENCE 1.31 230. ﬂuorescent GENOTYPE DNA Ladders provide a dual-ladder format to facilitate automated lane tracking and eliminate multiple gel loadings. H.70 369. Darﬂer.99 232. Jordan. 11728. The electropherograms illustrate the wide size range and even peak intensities of the ladders.77 174. All lanes were loaded simultaneously and the 2 distinct band patterns were visible. M.46 247. 11729).79 264.91 173.e l e c t r o p h o r e s i s RESULTS AND DISCUSSION The GENOTYPE TAMRA DNA Ladders were analyzed in the dual-ladder conﬁguration (ﬁgure 1). The expected size range was determined by Genethon from analysis of a large number of alleles for each locus.200 – 800 – 400 – 0– GENOTYPE TAMRA 50–500 DNA Ladder 1. error-free lane tracking by the software. Orientation markers (doublets and triplets) of the GENOTYPE DNA Ladders are indicated by arrows. Capillary electrophoresis of GENOTYPE TAMRA DNA Ladders.13 245.90 174.200 – 800 – 400 – 0– GENOTYPE TAMRA 60–500 DNA Ladder FIGURE 3. (1999) FOCUS 21. Markers were ampliﬁed using Genethon published sequences. Size (b) 50 – 100 – 150 – 200 – 250 – 300 – 350 – 400 – 450 – 500 – Fluorescence Intensity 1. EDITOR’S NOTE: The ladders are also available labeled with ROX (GENOTYPE ROX 50-500 DNA Ladder. The ladders accurately sized human DNA markers using gel-based or capillary-based (data not shown) electrophoresis systems.72 272.08 272. In summary. No. 1.46 230. No. J. Cat. and GENOTYPE ROX 60-500 DNA Ladder.82 264. Size determination of dinucleotide repeat microsatellite markers.05 273. The measured sizes calculated using the GENOTYPE DNA Ladders (table 1) were consistent with data obtained previously using commercially available ladders (data not shown).70 369. and Solus.72 TABLE 1.88 173. allowing automated. and the sizes of the amplicons were determined using the GENOTYPE TAMRA DNA Ladders on an ABI 377XL DNA Sequencer. Locus D1S196 D1S220 D1S234 D1S235 D9S167 D16S671 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 Expected Size Range (bp) 267–297 231–251 226–238 175–195 260–286 338–372 Calculated Size (bp) GENOTYPE TAMRA GENOTYPE TAMRA 50-500 DNA Ladder 60-500 DNA Ladder 272. The GENOTYPE TAMRA DNA Ladders were analyzed by capillary electrophoresis (ﬁgure 3).
and responsive to all types of inﬂammatory stimuli..5 54.1 58. According to the references.5 54. ELISA is used to measure cytokine induction. After 24 h. monocytes and macrophages are able to release mediators (e. TNF-α) accumulated stably in the culture medium. Applying an easy-to-use one-step RT-PCR procedure. The same volume of medium without LPS was added to control wells. Ekke Liehl.4 59. The cell lysates were harvested after scratching the well with a 200-µl pipette tip and multiple resuspensions. very mobile. Lysates of 6 wells were pooled in a 1.7 49.7. For ELISA assays. rapidly produced cytokines (e.6 58. Macrophages are active as phagocytes and produce mediators that attract other leukocytes and/or initiate reconstruction of tissue lesions. We show that the 2 methods produce corresponding and complementary firstname.lastname@example.org 54. the message data correlated with ELISA results on the release of the respective mediators. For RT-PCR analysis. Studying cytokine release from macrophages may help us understand fundamental mechanisms of inﬂammation.3 59.7 49. Another method to study the induction of a mediator is RT-PCR. primer sets were designed to amplify a DNA fragment from at least 2 exons of each target so that the fully spliced. one-step RT-PCR was easy to use and unfailing.p c r Monique Bongers. It is highly versatile. “s” is the sense primer and “as” is the antisense primer.com One-Step RT-PCR to Detect in Cytokine/Chemokine Induction Macrophages ABSTRACT The macrophage is a central cell in inﬂammation.0 ATG GGT CAG AAG GAT TCC TAT GTG CTT CAT GAG GTA GTC AGT CAG GTC CGC ACC TCC ACA TAG CTT ACA G CCT ATC CTG CCC ACG TGT TGA G GAC GAG ACC AGG AGA AAC AGG G AAC GGA GAA AGA AGA CAG ACT GCT TGG GTG GGA TGT AGC TAG TTC C AGT TTG CCT TGA CCC TGA AGC C GGA AAA ATG GAT CCA CAC CTT GC TCT CTT CCT CCA CCA CCA TGC AG GAA GAG TCC CTC GAT GTG GCT A CCC TTT TCT GTT CTG CTG ACA AG CCA CAA TAG CAG AGA AAC AGC AAT AAC CCC GAG CAA CAC CAT GAA G TCT CAT CAG TTC TAT GGC CC GGG AGT AGA CAA GGT ACA AC CTG GAC AAC ATA CTG CTA ACC GAC ATT CAT TCA TGG CCT TGT AGA CAC C GTT CTC TGG GAA ATC GTG GA TGT ACT CCA GGT AGC TAT GG TTG ACG GAC CCC AAA AGA TG AGA AGG TGC TCA TGT CCT CA ATG TGG CTG CAG AAT TTA CTT TTC CT TGG GCT TCC TCA TTT TTG GCC TGG T TABLE 1. e. with a special focus on chemokines.3 54.novartis. Based on previous experience. 5% CO2 in a humidiﬁed atmosphere. at a time when macrophages initiate the liberation of most of the mediators measured. Supernatants were discarded and 170 µl TRIZOL® Reagent were added per well. However. METHODS STIMULATION OF CELLS. 66 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 .0 54.. mature mRNA product could be distinguished from the unspliced mRNA or contaminating DNA products. mRNA data give additional information when used with ELISA. Cells were routinely passaged by scraping with a split ratio of 1:5. he macrophage plays an important role in immune responses (1). In most cases. cells were stimulated with 1 µg/ml LPS (Salmonella abortus equi) by adding the LPS Primer (Reference) β-actin-s β-actin-as IP-10-s (3) IP-10-as (3) KC-s (3) KC-as (3) MIP-2-s (3) MIP-2-as (3) MCP-1-s (3) MCP-1-as (3) MIP-1α-s (3) MIP-1α-as (3) MIP-1β-s (3) MIP-1β-as (3) TNF-α-s (4) TNF-α-as (4) IL-10-s (5) IL-10-as (5) IL-6-s (4) IL-6-as (4) IL-1β-s (4) IL-1β-as (4) GM-CSF-s (6) GM-CSF-as (6) Sequence T in 20 µl medium/well.8 54.7 macrophages (ATCC) were grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM) with 10% heat-inactivated FBS and penicillin/streptomycin at 37°C. Austria e-mail: monique.4 44. when only transcription of a mediator is induced but not translation.9 54. RAW cells were seeded at 2 × 105 cells/ml in 200 µl of medium/well on ﬂat-bottomed 96-well microtiter plates in the afternoon the day before stimulation..7 66. ELISAs require speciﬁc antibodies.1 56. The β-actin primers were designed by the author.9 54.2 51. which are not always available for newly discovered cytokines. The system is the widely used murine cell line RAW 264. Furthermore. and wound healing. since previous experience had shown that most mediators reached saturation at 24 h. supernatants were harvested and stored at –20°C. We applied one-step RT-PCR as well as ELISA in a simple in vitro system of macrophage cytokine/chemokine induction. stimulation was as described above.3 58.0 42. Total Product Size (bp) 359 431 530 466 582 561 540 231 300 227 223 435 Tm (°C) 56.g. cells were harvested 5 h after stimulation. 59 1235 Vienna. Murine RAW 264.0 57.g. the macrophage is central not only in host defense but also in wound healing (2). Also. Notably.6 57. We describe methods to study mediator production from the murine macrophage cell line RAW 264. RT-PCR primers. Particularly. and Johannes Barsig Novartis Research Institute Brunner Str.7 stimulated with the classical bacteria-derived stimulus lipopolysaccharide (LPS). cytokines) that activate and modulate other immune and non-immune cells.5-ml tube.g. but in 1 ml/well on 24-well plates. Importantly.3 57. host defense. The next morning. we demonstrate the rapid induction of a variety of cytokine/chemokine mRNAs by LPS in the macrophages.
A ﬁnal extension at 60°C for 7 min was performed. as a control. 30. Certiﬁed using an overnight coating. 1 h biotinylated antibody (then 4 wash steps). ELISA. as long as cycle numbers are controlled. and the difference between control and LPS stimulation was nearly gone. saturation was reached for LPS-stimulated cells. ONE-STEP RT-PCR. RNA was dissolved in 22 µl of nuclease-free water and stored at −70°C. Samples were taken from each tube after 20. One-step RT-PCR was used to investigate mRNA induction by LPS. Lane 2. After 20.05% Tween 20 in PBS). 2 µl loading buffer and 13 µl water were added to samples. and 10 µl were separated on a 2% agarose gel in 1X TBE with 0. Furthermore. β-actin products were analyzed. 50 pg/ml. Based on these results (and in accordance with the literature). Cells not treated with LPS. Bands were visualized with a GIBCO BRL® UV Transilluminator. and the data were analyzed using the Softmax Pro 2. 1 h blocking (10% FBS and 0. The graphs are densitometry of the gels.p c r 100 bp DNA Ladder β-actin 1 2 1 2 1 2 1 2 Control C 1 2 1 2 1 2 1 MIP-1α 2 1 2 C Control 1 2 RNA was isolated as recommended by the manufacturer.1. 30. and 35 cycles. 30 min Streptavidin-POD conjugate (then 5 wash steps). The ONE-STEP RT-PCR System can be used for semi-quantitative and even quantitative RT-PCR by adding internal controls and adjusting target signals to those. 60°C for 30 s. By 30 cycles. Brieﬂy. RESULTS AND DISCUSSION CYTOKINE/CHEMOKINE MRNA INDUCTION BY LPS. The lanes at 35C are controls done without addition of RT.5 mg/L ethidium bromide at 110 V. 94°C for 1 min. but at 25 cycles the unstimulated cells also had a signal.1 and 2. and 72°C for 60 s. Final images were composed using the Corel 7 software package. Lane 1. ODs between 0. First. and blank values were below OD 0. Detection limits for the ELISAs were 10 to F O C U S (1 9 9 9) V O L U M E 2 1 600 bp – 20 25 30 35 35C 20 25 30 35 35C 100 100 80 Signal Intensity [% max] Signal Intensity [% max] 80 60 –LPS 40 +LPS 60 40 20 20 0 20 25 30 Cycle Number 35 0 20 25 Cycle Number 30 35 FIGURE 1.5 were used by analyzing samples in the appropriate dilutions. Antibodies and recombinant cytokines were used at the concentrations recommended by the suppliers. These data indicate that comparable amounts of RNA were subjected to RT-PCR from control and LPS-incubated cells. 30-s pauses were programmed to obtain 5 µl of sample. and 10 to 30 min BM Blue incubation. For samples.7 cells analyzed by one-step RT-PCR. then 35 cycles of 94°C for 15 s. mRNA induction in RAW 264. this method is close to semi-quantitative.05% Tween 20® in PBS. the level of β-actin products was similar with and without LPS at all cycle numbers (ﬁgure 1). The amount of β-actin product increased with increasing cycle number and was saturated by 30 cycles. A standard ELISA was performed with NUNC Maxisorp F96 Immunoplates. As expected. and 35 cycles for β-actin and MIP-1α. Incubation was at 60°C for 30 min. 67 N U M B E R 3 . Control without RNA. 20 cycles showed induction of message in LPSstimulated cells. The importance of sampling at various cycle numbers was more evident with induction of MIP-1α (macrophage inﬂammatory protein-1α). 4 h sample incubation (then 4 wash steps). Cells treated with 1 µg/ml LPS. 25. RT-PCR was performed using the S UPER S CRIPT ™ O NE -S TEP RT-PCR System. 25.2. which agrees with the ELISA data below. The quantity of RNA was determined by A260. sampling from one tube during RT-PCR kept RNA consumption and costs low. Each plate contained a standard curve (10 ng/ml to 10 pg/ml in duplicate) and 2 blank wells with blocking buffer. then 2 wash steps with 0.1. one-step RT-PCR was performed on 1 µg RNA using 50 pmol each primer (table 1) in a 50-µl reaction. Lane C. The plates were read with a SPECTRAmax Reader. and images and densitometry analysis used the Kodak® Digital Science EDAS 120 System.
04 TABLE 2. FOCUS REFERENCES 1. 685. These enzymes have to be activated WITH LPS.6 ± 0.7 cells are useful to study in vitro cytokine/chemokine release from murine macrophages (except for KC). KC was not produced by the macrophages. 2. Immunol. L. possibly by a second signal. DiPietro. R.7 macrophages are useful to study induction of a variety of cytokines/ chemokines. Pharmacol. with some interesting exceptions. Mielke. Yan. Products are shown from cycle numbers where reactions behaved linearly (cycle numbers below mediator names).. note the high cycle number). Cytokine/chemokine production in RAW 264. 25.7 MIP-1β Control LPS-Stimulated 22 ± 2 183 ± 39 IL-6 nd 82 ± 2 MIP-2 1.. Immunol. themselves.. Mediator release from LPSstimulated macrophages was examined by ELISA. and RAW 264. 3016.2 ± 0. Yan. 5. and Milon. induction of KC mRNA and protein was seen in LPSstimulated murine bone marrow-derived macrophages (data not shown). 144. Oakes. 210.. 457. RAW 264. However. M. 175.. the chosen primers worked under these conditions.-R. J. nd = not detectable. Immunol. mRNA induction after LPS stimulation was investigated for selected cytokines/ chemokines.A.5 ± 4. 70...A. RT-PCR results of mRNA induction corresponded to the ELISA results on protein in the supernatant. Gordon.07 ± 0.01 MCP-1 0. X. Our protocol suggestions may speed up the work of other investigators interested in this area. except for KC (neutrophil-speciﬁc chemokine homologous to human GRO-α) (ﬁgure 2). M. unlike the frequently used peritoneal macrophages.. (1998) Res. 6. G. In most cases. the S UPER S CRIPT O NE -S TEP RT-PCR System provides a way for the non-molecular biologist to easily and rapidly obtain mRNA induction data.D. Meth. even though low but signiﬁcant mRNA was seen (see ﬁgure 2. and Hahn.88 ± 0. Falanga. 149. Singer. (1997) Toxicol. Appl. despite clear appearance of mRNA. They can be obtained at high number and purity. Controls without RNA or without RT were performed in each case and did not yield products (data not shown). and 35 cycles..01 ± 0. These mediators were induced by the treatment. and Lausch. In summary.. IL-10 was not upregulated by LPS. Y. LPS induced the production of most of the mediators analyzed (table 2). 4. Most of the cytokine messages were constitutively expressed at low levels in unstimulated cells and were highly upregulated by LPS.02 0. Induction of mRNA does not always mean that the protein is produced and released. 3.7 cells. these methods are more demanding in terms of equipment and experience of personnel compared to the procedures described here.7 cells (not in the table). Rev. However. Blankenstein. Colle. These experiments show that one-step RT-PCR is highly sensitive. Hevin. T. IL-10 released at a low level immediately bound in an autocrine manner to receptors on the cells. IL-1β was not released from the RAW 264. H. and the cells are rather quiescent when not stimulated. Samples were taken from each tube after 20. (1992) J. P. Cells treated with 1 µg/ml LPS.4 55. (1996) J. Lane 2. 16. Also. J. 149.4 13 ± 9 IL-10 0. Ehlers. S. Dinarello. Data are mean ± S. Also.E.7 cells analyzed by one-step RT-PCR.8 ± 0.2 103 ± 33 GM-CSF nd 0. Cells not treated with LPS. (1997) J.7 ± 0. 1277. Zhou. Lane 1.B.03 ± 0.0 ± 1. 294.-T.-H. This can be explained by the complex mechanisms of IL-1β release from cells (7). All PCR products had the predicted sizes. 68 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 ..-H..J. CYTOKINE/CHEMOKINE RELEASE AFTER STIMULATION 600 bp – MIP-2 25 KC 35 MCP-1 25 IP-10 25 MIP-1β 25 IL-10 35 TNF-α 20 IL-6 35 IL-1β 35 GM-CSF 35 FIGURE 2. Possibly. C. The nascent protein lacks a signal sequence and is produced as a precursor that has to be processed by IL-1 converting enzymes. and Pestka. D. (1995) Shock 4. 7. J. mRNA induction in RAW 264. in ng/ml for N = 3.6 IP-10 1.05 ± 0.09 ± 0. Su. H. Immunol. as seen with RT-PCR. S. (1998) Int. Virol.p c r One-Step RT-PCR continued 100 bp DNA Ladder 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 or by using kinetic methods to produce quantitative results.01 KC 0. 233. thus prohibiting detection by ELISA of supernatants. which may hence be more suitable to start with. B. 30.9 60 ± 8 MIP-1α 7.01 9. TNF-α Control LPS-Stimulated 0.01 0.
the rest are <25 bases.01 97. or HPLC puriﬁcation. This trityl group protects the nucleotide from undergoing unwanted chemical reactions during the synthesis cycle and is removed immediately before a new nucleotide is added. the coupling efﬁciency would be 100%. Why is coupling efﬁciency important? It is used to determine the amount of full-length oligonucleotide produced. Coupling efﬁciency affects the amount of full-length primer. Why is trityl group analysis important to perform? TABLE 1. • COUPLING—The next nucleotide is added to the reaction and couples (covalently attaches) to the ﬁrst nucleotide.71 61. where n = total number of nucleotides. • DEBLOCKING—The 5´-trityl group is removed from the second nucleotide to prepare it for further cycles.66 37. the theoretical yield for a 25-mer would be: 0. How do I determine the percentage of full-length oligonucleotide? Every nucleotide added during DNA synthesis has a dimethoxy trityl (trityl) protecting group attached. Table 2 gives guidelines for the minimum purity for a range of applications.9924 = 0.16 14. PAGE. • CAPPING—Any of the ﬁrst nucleotide that failed to react is capped so that it will not play a part in the subsequent synthesis cycles. attached to the solid support via a chemical linker arm.61 74. is deprotected by removing the trityl-protecting group.79 The trityl group is colorless when attached to a DNA base but gives a characteristic orange color once removed. The ﬁrst nucleotide is attached to a solid support to anchor the growing DNA chain in the reaction column. ∼79% of the oligonucleotide molecules in the tube are 25 bases long. A chain of nucleotides is built by repeating the synthesis cycle until the desired length is achieved.p c r : Technical Services Part of the Technical Support and Training Team Life Technologies.03 91. The intensity of this color is measured by spectrophotometry and is directly related to the number of trityl molecules present. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge. synthesis typically can reach ∼99%. Inc. hence.12 55. To calculate the percentage of full-length oligonucleotide.87 98% Coupling 98 96. We use continuous trityl analysis to measure coupling efﬁciency throughout the synthesis. If every available nucleotide on the DNA chain reacted successfully with the new nucleotide. the coupling efﬁciency. For example.12 83. How do you measure coupling efﬁciency? The percentage of full-length oligonucleotide depends on the coupling efﬁciency of the chemical synthesis. use the formula: % full length= (coupling efﬁciency)n-1. Maryland 20849 h e l p f u l t i p s Helpful Tips for Custom Primers Oligonucleotide Length Percent Full Length 99% Coupling 2 3 4 10 20 30 50 95 99 98. Rockville.04 94. N U M B E R 3 69 . it may be necessary to remove the oligonucleotide that is not full length. Table 1 shows the effect of a 1% difference in coupling efﬁciency and how this inﬂuences the amount of full-length product for different-length oligonucleotides. Depending on the application and oligonucleotide length. DNA synthesis consists of a series of standard β-cyanoethyl chemical reactions. even a small difference greatly affects the amount of full-length product.98 Oligonucleotides are made using computer-controlled DNA synthesizer and the patented parallel array synthesis method. Coupling efﬁciency for DNA F O C U S (1 9 9 9) V O L U M E 2 1 Therefore. No chemical reaction is 100% efﬁcient.37 68.11 38. Each cycle results in the addition of a single nucleotide. This means that at every coupling step approximately 1% of the available nucleotides fail to react. What is coupling efﬁciency? H ow are your oligonucleotides made? Is a small difference in coupling efﬁciency important? Since coupling efﬁciency is cumulative during DNA synthesis. Each cycle consists of: • DEBLOCKING—The ﬁrst nucleotide. it is possible to calculate the percentage of nucleotides coupling successfully and. The average efﬁciency is close to 99%. By comparing the absorbance of trityl throughout synthesis.35 82. • OXIDATION—The bond between the ﬁrst nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain. Why do oligonucleotides sometimes require puriﬁcation? Coupling efficiency measures how efﬁciently the DNA synthesizer added new nucleotides to the growing DNA chain. This produces a free 5´-hydroxyl group to react with the next nucleotide.
GC content. Losses occur during processing. approximately 50 nmoles of the ﬁrst nucleotide are added to the DNA synthesizer.g.1 to 0. RNA polymerase promoters) First-strand cDNA synthesis for RT-PCR Fluorescent sequencing Cycle sequencing Isothermal sequencing Site-directed mutagenesis First-strand cDNA synthesis for generation of libraries GENETRAPPER® screening Production of cloning adapters Gel shift assays AFLP™ analysis CFLP™ analysis Antisense TABLE 2. Primer Size (nucleotides) 10–20 20–30 30–40 40–50 50–60 Expected OD 4–7 7-9 9–10 10 10 Expected nmole 40–32 32–27 27–22 22–18 18–15 Take the A260 reading by diluting 10 µl of the oligonucleotide with 990 µl of water (1:100 dilution).p c r : h e l p f u l t i p s Helpful Tips for Custom Primers continued Application PCR PCR using primers with critical 5´ sequences (e. and quality control. why don’t I receive 50 nmoles of product? Synthesis scales refer to the amount of starting material present. not the amount of product produced.000 PCRs (for 100-µl reactions using primer concentration of 0.9 nmole/OD.5 µM). and coupling efﬁciencies can vary the yield of product. This oligonucleotide has an extinction coefﬁcient of 4. Oligonucleotide length. transfer of material. On average. 100% recovery.14 OD. Suggested primer purity. TE is recommended over deionized water since the pH of the water is often slightly acidic and can cause hydrolysis of the oligonucleotide.. the 50-nmole scale will supply enough oligonucleotide for 1. When a 50-nmole scale synthesis is speciﬁed.0). 70 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . 1 mM EDTA].14 OD/ml × 100 × 4. Primer length affects yield. sequence. restriction endonuclease sites. The A260 value is 0. For 24 nmole of an oligonucleotide: Volume = [24 × (1/103)] × 106 µl 100 µmol/L L = 240 µl How do I reconstitute my oligonucleotide? Dissolve the oligonucleotide in TE [10 mM Tris-HCl (pH 8. Puriﬁcation of the oligonucleotide also affects ﬁnal product yield as determined by ﬁnal OD. Primer length matters Calculating primer concentration To calculate the concentration of an oligonucleotide dissolved in 1 ml: Concentration = A260 × Dilution Factor × Extinction Coefﬁcient × Conversion Factors The length of the primer affects the yield. The table assumes 98% coupling efﬁciency. as seen in table 3. This is the same for all manufacturers of synthetic DNA. Concentration = 0. and 50% GC content using the 50-nmole scale.000 to 5. Minimum Suggested Purity Standard Cartridge Standard Standard Standard Desalted Cartridge Cartridge PAGE Cartridge Cartridge Standard Desalted HPLC puriﬁcation is cited most frequently When I order the 50-nmole scale.9 nmole/OD × 1 µmol/103 nmole × 103 ml/L = 69 µM To calculate volume to dissolve an oligonucleotide in for a 100-µM solution: Volume = Number of nmoles × (1 µmol/103 nmole) Desired Concentration × Conversion Factor (for L to µl) TABLE 3.
D. The oligonucleotide dissolved in water is stable for at least 6 months at –20°C in the absence of nucleases. John Wiley and Sons.55. To learn the latest in PCR techniques. Speciﬁcity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures. NewYork. • Avoid mismatches with the target at the 3´ end. Guidelines for primer design for PCR An important parameter for primers is the melting temperature Tm. Moore. (1998) FOCUS 20. see Fox. The ability of ethidium bromide to stain oligonucleotides is poor and variable depending on sequence and composition. A.R. • Avoid sequences with the potential to form internal secondary structure.. Properties that are sequence dependent in oligonucleotides include extinction coefﬁcient and A260/280 ratio. J. • Avoid a GC-rich 3´ end. Do not store oligonucleotides in water at 4°C. (1995) PCR Essential Data.D.. • Avoid complementary sequences at the 3´ end of primer pairs. The following guidelines describe the desirable characteristics of a primer sequence: • Typical primers are 18 to 24 nucleotides. Inc. Be sure the water used is at neutral pH to avoid depurination. • Select primers that are 40% to 60% GC or mirror the GC content of the template. the A260/280 ratio is not an indicator of purity because the ratio of a given oligonucleotide is highly sequence dependent. calculate the extinction coefﬁcient using an equation that incorporates the contribution of each base and the effect of base stacking [Newton. Oligonucleotides of the same size can migrate differently in denaturing polyacrylamide gels. J. Seidman. • For accurate determination of oligonucleotide concentration. (1999) FOCUS 21. comparison of an oligonucleotide to a molecular size standard or another oligonucleotide of known size is not a reliable way to determine its size. The Tm is necessary to establish an annealing temperature for PCR.. A.G. P. Poorly designed primers may amplify other. Natarajan. [Ausubel. Analysis of oligonucleotides by electrophoresis Analysis of oligonucleotides by absorbance spectroscopy Annealing temperature for PCR primers Unlike large DNA molecules. enroll in one of our PCR Techniques or Advanced PCR Techniques courses at the Life Technologies Training Center. K. and Fox. Brent. For more information. • Design primers with G or C residues in the 5´ and central regions. This is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. For more information. R. Kingston. and these differences are composition and sequence dependent. D.] • For oligonucleotides. P. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 71 .] Use of ethidium bromide for detection and quantitation of an oligonucleotide is not reliable. See inside front cover for the schedule.M.. sequence can affect the migration rate of oligonucleotides. FOCUS The ideal PCR primer pair anneals to unique sequences that ﬂank the target and not to other sequences in the sample. The oligonucleotide dissolved in TE is stable for at least 6 months at –20°C or 4°C. and Struhl. 84. New York. • Addition of 40% formamide (in addition to 8 M urea) to the acrylamide gel solution removes most sequence dependent migration effects. The reason for this is the large differences in extinction coefﬁcients of individual nucleotides.. New York.K. Since most formulas provide an estimated Tm value.E.. New York.p c r : h e l p f u l t i p s How long can I store the oligonucleotide? The lyophilized oligonucleotide is stable at –20°C for at least 1 year. John Wiley and Sons. Annealing temperatures are generally about 5°C below the Tm of the primers. Reasonable annealing temperatures range from 55°C to 70°C. R. D. p. nontarget sequences. Smith. • Since migration is sequence dependent. 2. F. (1994) Current Protocols in Molecular Biology. see Sewall. the annealing temperature is only a starting point...
Santiago. The DNA was ligated to a vector and transformed into DH5αF´™ competent cells. Manuel Lladser. and incubated overnight at 37°C. female. After centrifugation at 12. since 1993. Vienna-60 (a white pupae temperature-sensitive mutant of S-6). Both strands of the clones were sequenced with the GIBCO BRL dsDNA Cycle Sequencing System. ﬁsh (8).P. Sequences were analyzed using the BLAST program from NCBI. capitata has been studied using multilocus enzyme electrophoresis and RAPDs (11−13). the DNA was vacuum dried and dissolved in 20 µl of sterile double-distilled water.000 miles of border is needed.1 M 2-mercaptoethanol] with 25 µl proteinase K (10 mg/ml) until the solution turned a reddish color. The DNA was ethanol precipitated. The DNA precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of cold ethanol.E. AFLP products were generated using the GIBCO BRL® AFLP Analysis System I according to the manufacturer’s instructions.a g r i c u l t u r a l b i o t e c h n o l o g y Gino Corsini. In this study. respectively). since 1996. 50 mM EDTA. Chile AFLP ™Analysis of the Fruit Fly Ceratitis capitata hile Chile was declared free of the med ﬂy in 1995. C.I. 1 2 3 4 M 5 6 FIGURE 1. The DNA was collected by centrifugation. Santiago. Each individual conserved in 95% ethanol at −20°C was dried under vacuum in a 1. The solution was collected and ethanol precipitated at −20°C for 30 min. DNA EXTRACTION . C.700 −1. F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . 3% w/v SDS. and a ﬁnal soak at 4°C. Strain S-6 (larva). Polymorphism of wild populations of C. TE was added to 400 µl. dried.uchile. 0. and the mixture incubated for 10 min at 65°C. Chile email@example.com)]. 72°C for 1 min. Strain Toliman (males). and Lluta were obtained from Centro de Producción de Insectos Estériles. SAG) located in Lluta. digested with EcoR I (lane 5) or undigested (lane 6). After electrophoresis. Lane M. and the pellet was washed with 70% ethanol.E. PCR was 35 cycles of 94°C for 30 s. The AFLP products (5 µl) were separated on 5. fungi (6).P. Lluta 72 strain is a cross of native ﬂies collected in valleys of southern Peru and Azapa (northern Chile) and has been reared at C. and dissolved in 50 µl TE buffer [10 mM Tris-HCl.000 × g.0). Toliman. dried. and dissolved in sterile water. Lane 4. After centrifugation. dried. so surveillance of over 2. GIBCO BRL Taq DNA Polymerase was used for PCR. and larva.000 × g for 20 min.I. One report related to AFLP analysis of arthropod species has been published (10). Augusto Manubens. 1 mM EDTA (pH 8.8% agarose TBE gels. 0. W METHODS MATERIALS. Lanes 5 and 6. and humans (9). the AFLP technique was used to detect genetic polymorphism in C.8% or 6. DNA was extracted from individual adult ﬂies or larvae from different strains. native populations of Ceratitis capitata remain in neighboring countries. Puriﬁed genomic DNA. DNA was incubated with 10 µl RNase A (10 mg/ml) for 45 min at 37°C and then treated with 10 µl of proteinase K (10 mg/ml) for 15 min at 50°C. ISOLATION AND CLONING OF POLYMORPHIC GENETIC The selected bands were cut directly from the dry gel. 56°C for 30 s. AFLP REACTIONS.E. submerged in TE buffer containing 1 M NaCl. the supernatant was extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). Lanes 1–3. and dissolved in 40 µl of TE buffer. the gel was dried and exposed to a Kodak X-Omat AR ﬁlm.5-ml tube and ground at room temperature in 200 µl of lysis buffer [50 mM Tris-HCl (pH 8.6 volumes of cold isopropanol was added to the supernatant and incubated at −20°C for 20 min. and the pellet was washed with 70% ethanol. MARKERS. and 1/4 of the samples were electrophoresed on 0.P. capitata (Wiedemann) (Diptera: Tephritidae). The AFLP technique was developed primarily to reveal the differences between cultivars of plant species (1) and has been applied to several crops. and the mixture was incubated on ice for 10 min. Sergio Lobos.I.cl Carlos Lobos Programa Nacional de la Mosca de la Fruta Departamento de Protección Agrícola Servicio Agrícola y Ganadero (Agriculture and Livestock Service) Bulnes 140. Genomic DNA was prepared from individual larvae or adult specimens. capitata from strains Seibersdorf-6096 (S-6). Ampliﬁed Fragment Length Polymorphism (AFLP) analysis is an efﬁcient DNA ﬁngerprinting method based on selective ampliﬁcation using PCR of restriction fragments from a total digest of genomic DNA. AFLP analysis has identiﬁed a larger number of molecular markers than RAPDs and RFLP in soybean (2) and cotton (3). from the Servicio Agrícola y Ganadero. Strain Lluta (male. and Daniela Seelenfreund Department of Biochemistry and Molecular Biology Facultad de Ciencias Químicas y Farmacéuticas Universidad de Chile Casilla 174 correo 22. Toliman is a cross between a native strain from Guatemala (Toliman) and the temperature-sensitive strain Vienna-42 that has been reared at C.dic. the DNA pellet was washed in 70% ethanol. The ampliﬁed DNA was visualized on a 3% agarose gel stained with ethidium bromide. 100 µl of 5 M potassium acetate were added.800 V) in 1X TBE on a Model S2001 gel electrophoresis system. and the sample was extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). Identiﬁcation of the genetic origin of accidentally introduced individuals would help to localize control and facilitate the surveillance. Chile. among other crops. The AFLP technique has been used to analyze genetic polymorphism of nematodes (4−5). After centrifugation for 10 min at 12. λ DNA/Hind III fragments. The sample was ampliﬁed using the same pair of primers selected from the AFLP reaction. corals (7).0% (w/v) acrylamide gels at 70 to 80 W (1. Another 200 µl of lysis buffer were added.
Biol. 156.R.. G.. 119. J. FIGURE 3. (1996) Plant Mol. primer pairs produced 35 to 50 bands from 75 to 500 bp. G.F. W. S-6 presented a characteristic marker with E-AGG/M-CAG primers (data not shown). Lluta. T. G. but few primer pairs produced distinctive patterns. Sonvico. Strain Vienna 60. M. Immunol. Schreiner. N U M B E R 3 73 .. and Zabeau. 80. Lin. Bleeker.. Vos. Lane 6. X. we have isolated and cloned the genetic marker bands from Vienna-60. the expected common marker band was seen with both primer pairs.. 11. Mueller. Kuiper.A. Beard. E.G. Li. For example. P.R. A. Rehner.. and F O C U S (1 9 9 9) V O L U M E 2 1 bp – 400 – 300 – 200 – 125 FIGURE 2. 13. J. V.. Otsen. Rep. 5. (1996) J.... J.. Strains were analyzed using the selective primer pair E-AAG/M-CAT. Saunders. 244. The primer pair E-AAG/M-CAT identiﬁed speciﬁc markers of Lluta. 260. L.. 95. R. M.E.. and Soliman. Ma. (1997) Focus 19. S. Manso. All other lanes show DNA extracted from adult ﬂies. M. and Zebitz. and Dunham. The AFLP patterns from several DNA samples of the same individual were reproducible (data not shown).. Ecol. Acid Res. 1208. Helminthol.H.A. Gugliemino.E. (1998) J.. and Matthews. A. B. 425. However. Erne. A. M. comparing individuals from the Lluta strain. Pot. (1998) Mol. Hogers. 258. Malacrida. Bandi.. A. 12.. R. S. Lane 3. D. Kuo. and Lenstra. a number of bands reﬂecting individual differences (and high genetic diversity) were observed. C. L.. Ude.. Arrows indicate the presence of the characteristic marker band. Liu.. Lane 5.. Bull. M. 93. Wajnberg. 291. Econ. PCR and Southern hybridization are in progress to verify that these marker bands can be used as speciﬁc probes. 6.. However.V. P. 14. The feasibility of isolating speciﬁc markers for each strain could eventually facilitate the analysis and monitoring of ﬂy populations. A. D.. Biol. we have applied the AFLP technique to identify laboratory strains of med ﬂies. Wolpl. Feng. and Vienna-60 in both male and female individuals (ﬁgure 2).S.. Z. (1996) J. Lane 4. Universidad de Chile. For each strain. P.P. MacDonald. S.. Hoekstra. Kersanach. K.. Barufﬁ. M. Ecol. T.. Therefore. Kenworthy. Arrows indicate characteristic marker bands for each strain using this primer pair. Strain Lluta. J. 23.P.S.. (1995) Nucl. Lipari. Toliman.. 360 bp (Vienna 60).... Van der Lee. Strain Toliman. Hornes.. Peleman.. Nichols.A. J. Generally. and Quesada-Allue. J. (1994) Genome 37. 9. (1996) Mol. All fragments were A/T rich (data not shown). 72. Dalmasso. R. 10. and Milgroom. Lanes 1 and 2 correspond to the analysis of larval tissue from strains S-6 and Lluta. (1995) Heredity 74. 4407.. J.. Reijans.a g r i c u l t u r a l b i o t e c h n o l o g y 25 bp DNA Ladder 25 bp DNA Ladder 3 4 5 6 A 1 2 3 4 5 6 7 8 9 10 B 1 2 3 4 5 6 7 8 9 10 1 2 & ( & ( & (&( RESULTS AND DISCUSSION Puriﬁed genomic DNA from an adult ﬂy or larvae yielded 4 to 5 µg of unsheared DNA with a 260/280 ratio of 1.. 7. Abad. Lopez. Our goal is to map the genetic diversity of native wild med ﬂy populations from different geographical locations surrounding the Chilean borders. even though this strain has been reared in the laboratory for >30 generations.. P. A. respectively. Semblat. (1998) Mol. Gen. 8. To use the marker bands as speciﬁc probes. and Gasperi. Plas. Preliminary analysis of wild populations from different geographical locations suggests that local patterns can be obtained. Genet. Haymer. M.O. M. ﬂies belonging to the same strain exhibited a number of bands that varied between individuals. M.. Maccari. and Knowlton. The fragments were 280 bp (Lluta).J.G. 2.. and Castagnone-Sereno. Comparison of AFLP patterns. (1998) Insect Mol. Strain S-6. and Toliman. Presence of marker band in strain Lluta individuals. F. Entomol. our analysis was focused on identifying marker bands that were common for all individuals of a speciﬁc strain.A. Reineke. A. Kinzler. ACKNOWLEDGEMENTS This work was ﬁnanced by the joint project FAC-SAG/PA-001/97 from the Facultad de Ciencias Químicas y Farmacéuticas. J. and the presence of the respective marker bands was detected for Toliman and Vienna-60 (data not shown). M. and Wiesneth. In summary. 7. 7. C. Prochnow-Calzia..N. 196. Methods 196.. P. FOCUS REFERENCES 1. R. 2 similar strains were screened with 64 primer combinations for at least one differentiating band (data not shown). 11. as well as a similarity in the banding pattern (ﬁgure 3).8 (ﬁgure 1). I. 5 males (lanes 1−5) and 5 females (lanes 6−10) were analyzed with E-AAG/M-CAT (panel A) and E-AAC/M-CAA (panel B). H. A search of GENBANK for these fragments did not match with sequenced genes from Drosophila or Ceratitis..6 to 1... 3. Saha. C. 5. Roos. Based on the initial screening. 8 primer pairs were chosen to analyze both sexes of 4 laboratory strains.. Damiani.A. N. Frijters.. 4. AFLP patterns from one male (() and one female (&) are shown.H. H. 119.. B. and the Servicio Agrícola y Ganadero. J. 89. These assays were performed for the other strains.. In initial AFLP experiments. M. Karlovsky. 210 bp (Toliman). (1999) Biol. and McInnis.. E..
F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . or 10. REACT buffers 1. the accumulated pyrophosphate from the PCR would further reduce the kinase activity. Teresa Myers. Additionally. serve as a substrate for the kinase. At best.5). however. T4 polynucleotide kinase would show partial activity. we do not suggest adding restriction endonucleases directly to PCR products. we know many researchers perform double digests to save time. However. Ampliﬁcation Grade DNase I does come with a 10X reaction buffer [200 mM Tris-HCl (pH 8. The reaction must be incubated for at least 1 h. 15]. 10 units of T4 DNA polymerase are added directly to the second-strand synthesis reaction. Rockville. it describes a protocol where calf intestinal alkaline phosphatase can be added directly at the beginning of a restriction digest using the REACT buffers mentioned above. however. 5 mM ATP. Technical Bulletin 18009-1 describes the activity of calf intestinal alkaline phosphatase. in all of the SUPERSCRIPT® Systems for cDNA Synthesis. 2. 10 mM (NH4)SO4.3). If the objective. The MgCl2 is lower than the suggested optimum of 10 mM. We do not recommend double digests because of possible buffer incompatibility problems and enzyme steric hindrance problems. Inc. DNase I does not come with a buffer. This piece addresses many commonly asked questions related to buffer compatibility. 100% enzyme activity is seen in 74 Although common PCR buffer [TrisHCl (pH 8. the best approach would be to clean up the PCR using a product such as the CONCERT™ Rapid PCR Puriﬁcation System prior to the digestion [see FOCUS (1999) 21. Maryland 20849 Buffer Compatibility for Common Reactions S aving time and reagents are objectives shared by many molecular biologists. and KCl) would support restriction endonuclease activity. The REACT buffers lack the ATP necessary for the ligase reaction. 25% polyethylene glycol].4 to 8. What is the activity of restriction endonucleases in PCR buffer? Yes. Taq DNA polymerase is still active at these temperatures. The buffers of the specialized PCR enzymes like E LONG ASE ® Enzyme Mix and PLATINUM® Pfx DNA Polymerase have not been tested with DNase I.15 mM β-NAD+. This means that 5´ overhangs generated by restriction endonucleases will be ﬁlled in by the Taq DNA polymerase. If enough of each enzyme (1 unit. In fact. 100 mM KCl. 5 mM MgCl2. None of the REACT buffers are comparable to the ligase buffer. and 20 mM MgCl2]. The composition of second-strand synthesis reaction is 25 mM Tris-HCl (pH 7. Can T4 DNA polymerase be added to the second-strand cDNA synthesis reaction to polish the cDNA ends? Yes. this approach is strongly discouraged. Choose the combination of buffers that yields the highest activity for both enzymes. If 2 enzymes can be used in the same reaction buffer.4). It is possible to use a REACT buffer that gives less than 100% enzyme activity for a particular restriction endonuclease and increase the amount of enzyme used in the reaction. The 5X T4 DNA Ligase Buffer is available separately. and Julie Brent Technical Services Part of the Technical Support and Training Team Life Technologies. so the Taq 10X PCR buffer is an alternative. Therefore. it could mean using both enzymes at the same time or at the very least it could save the time and resources associated with puriﬁcation between the reactions. 4. Even though restriction endonuclease reactions are done at lower temperatures than Taq DNA polymerase reactions. Can T4 DNA ligase be used in any REACT buffer? Can you use a PCR buffer with DNase I? How do I choose a REACT® buffer for my double digest with restriction endonucleases? GIBCO BRL Restriction Endonucleases are provided with a REACT Buffer to ensure optimal performance.6). 6. The pH of most PCR buffers is slightly above the optimum pH range of 7. Since most primers are purchased without 5´ phosphates. can T4 polynucleotide kinase be added to PCR? Is there a simpliﬁed protocol for using alkaline phosphatase that does not require phenol extraction and ethanol precipitation after restriction endonuclease digestion? Yes. The Taq 10X PCR buffer and the Ampliﬁcation Grade DNase I buffer have the same formulation. or search for double digests in the TECH-ONLINE section of our web site. In fact. 150 units. the use of TAQUENCH™ PCR Cloning Enhancer will inhibit the Taq DNA polymerase activity during the restriction digestion [see FOCUS (1998) 20. MgCl2.m o l e c u l a r b i o l o g y : h e l p f u l t i p s Joe Crouse. 500 mM KCl. and 1 unit. 11]. respectively) is used. The bulletin can be found in the TECH-ONLINE section of our web site in the Molecular Biology manuals and technical bulletin section.0 for the forward kinase reaction. Enzyme buffer activity charts can be found on pages R-52 and R-53 in the 2000–2001 GIBCO BRL Catalogue. 5 mM DTT. and temperature-sensitive alkaline phosphatase in 6 common restriction endonuclease buffers.2 mM DTT. 0. and 1. Additionally. The remaining dNTPs from the PCR could. Alternatively. bacterial alkaline phosphatase. 3. 50 mM MgCl2. T4 DNA ligase comes with a 5X T4 DNA ligase buffer [250 mM Tris-HCl (pH 7. ® SM No. is to get signiﬁcant phosphorylation.
The ligation of the adapters is done in 1X buffer at 16°C.” F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 75 . In the SUPERSCRIPT Choice System for cDNA Synthesis.m o l e c u l a r b i o l o g y : h e l p f u l t i p s Are any of my REACT buffers compatible with the Klenow (Large Fragment DNA Polymerase I)? Klenow buffer is in essence REACT 2 buffer 10X [500 mM Tris-HCl (pH 8. T4 polynucleotide kinase is added to this reaction for the kinase step. To ﬁnd out how active your restriction endonuclease is in REACT 2 buffer. This means that once the fragment is ligated to the adapters. 5 mM ATP] is recommended for the adapter ligation reaction and the phosphorylation reaction. Before polishing the ends of a cut fragment. 50 mM MgCl2. After heat inactivating the T4 DNA ligase at 70°C for 10 min. I like to think of myself as a Ricket Scientist. check the reference section in the Life Technologies 2000–2001 Catalogue (page R-52). T4 polynucleotide kinase will show some activity in PEG-containing T4 DNA ligase buffers. however. For enzymes that are not heat sensitive. the use of a unique 5X Adapter Buffer [330 mM Tris-HCl (pH 7. when cloning adapters are added to fragments to be subcloned. heat inactivate the restriction endonuclease digestion for 10 to 15 min at 65°C. and 500 mM NaCl]. only one end is phosphorylated. FOCUS “I study the biomolecular mechanisms of vitamin C deﬁciency diseases. 5´ phosphates will need to be added to the resulting product. 100 MgCl2.6). do a phenol-chloroform extraction. What is the best buffer to use to avoid an ethanol precipitation step between a ligation and kinase reaction? Oftentimes.0).
with higher levels of bicarbonate. Pyruvate can be oxidized further to produce energy and CO2 or converted to lactate.c e l l c u l t u r e : h e l p f u l t i p s p l a n t b i o t e c h n o l o g y How Basal Media Provide theCulture Optimal Environment for Cell o maximize success. primarily D-glucose. The growth-promoting factors present in serum provide a “mechanism” to easily adapt cells from one formulation to another. Traditionally. 76 F O C U S (1 9 9 9) V O L U M E 2 1 N U M B E R 3 . and the amino acid L-glutamine are the major carbon and energy sources in media. Serum is largely undeﬁned. if cell density is low or cells have entered into a lag phase. media like D-MEM (3. and vitamins. and cell attachment. irrespective of cell type. Inc.7 g/L). Vitamin concentration is important in terms of cell survival and growth rate rather than directly affecting maximum cell density. Vitamins are precursors of enzymatic cofactors. To counter this. For most applications. Cell culture medium maintains pH and osmolality essential for viability and provides the nutrients and energy required for cell growth. A signiﬁcant portion of the energy required to maintain cell growth (∼30% of the energy produced. but it supplies essential growth factors and hormones required for cell growth. require ∼10% CO2 to maintain correct pH. Typical ranges for L-glutamine in formulations are 1 to 4 mM. basal cell culture media have been buffered by bicarbonate (HCO3). However. More specialized medium formulations. L-alanyl-L-glutamine. Serum also provides an additional source of carbohydrates. The high L-glutamine concentration present in formulations may also be due to its somewhat unstable nature in solution. amino acids. These dipeptides are extremely stable in solution and can be autoclaved without decomposition.5 g/L. membrane potential. Therefore. due to L-glutamine’s importance as an energy source as well as its role in protein synthesis. this can vary with cell type) comes from the oxidation of L-glutamine. In carbohydrate catabolism (glycolysis). FOCUS To learn more about cell culture. bicarbonate-buffered media require the use of incubators with an atmosphere of 5% to 10% CO2. Basal cell culture media minimally consist of salts. vitamins. A slow-growing cell line (long doubling time) will grow in low or high glucose. At a minimum. basal medium must contain the essential amino acids— those amino acids that cannot be synthesized at a rate adequate to meet metabolic requirements. and nutrition. they may not produce sufﬁcient CO2 to maintain optimal pH. Exposure to lower-than-optimal glucose levels could induce the cells to enter a lag phase. glycyl-L-glutamine) can be substituted for L-glutamine in medium. require ∼5% CO2. CO2 is evolved. ENERGY SOURCES Carbohydrates. such as MEM (1. However.5 to 2. ∼10 times the concentration of other amino acids. This paper discusses the functions of these components of basal media. Kevin Grady Technical Services Part of the Technical Support and Training Team Life Technologies. Cultured cells have two primary paths of energy (ATP) production: conversion of glucose to lactate (or full oxidation to CO2) and oxidation of L-glutamine to CO2 and ammonia. amino acids. The important factor is using the correct percent CO2 based upon a medium’s bicarbonate level to maintain physiological pH. a fast-growing cell line requires higher glucose levels to maintain its metabolic rate. D-glucose is converted via several reactions to pyruvate. and vitamins. The dissolved CO2 forms a buffering system with the bicarbonate. enroll in our Cell Culture Techniques or Advanced Cell Culture Techniques courses at the Life Technologies Training Center. developed for low serum supplementation or high cell density. choice of basal medium may be secondary to using the “right” serum. pH. osmolality. and energy sources. To counter these potential problems. As cells grow. basal media generally require serum supplementation. Grand Island. often have non-essential amino acids added to ensure that amino acids do not limit the maximum cell concentration attainable. serum supplementation lessens the concerns about a basal medium choice and whether a speciﬁc formulation provides the essential levels of carbohydrates. the in vitro culture conditions are set up to mimic in vivo conditions of temperature. enzyme cofactors. In a serum-supplemented application. See inside front cover for the schedule. Media with low levels of bicarbonate. Culture media are buffered to compensate for the cellular production of CO2 and lactic acid as metabolic by-products.2 g/L). the metabolic rate of a cell line correlates to the optimal glucose level. a dipeptide form of L-glutamine (GLUTAMAX ™ I. New York 14072 T PH AND OSMOTIC PRESSURE The salts in basal medium are primarily responsible for providing physiological pH and osmotic pressure. NUTRIENTS Amino acids are incorporated into proteins. Another energy/carbon source that is present in media is pyruvate. Traditional glucose levels in culture media range from 1 to 4. Vitamins are needed for cell growth and multiplication. GLUTAMAX II. amino acids. Generally.
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