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Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy

Mini Kit
©DGC, Indiana University- October 11, 2007

Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN
RNeasy Mini Kit
Includes protocol for RNA Quantitative and Qualitative Assessment
Jacqueline Ann Lopez and Elizabeth Bohuski
Based on DGRC protocol by Kevin Bogart, Jason Conaty, and Justen Andrews
INTRODUCTION

The utilization of microarrays for transcription analysis, cDNA synthesis, Q-RT PCR, and Northern
Analysis requires firstly the extraction of RNA from a biological source of interest. We describe this
procedure in detail along with RNA quality control measures. As per all steps involved in RNA isolation, it
is essential to avoid latex gloves – use only nitrile gloves. It is likewise important to guard against sources of
dust and nucleases. We recommend using barrier pipette tips, and filtering all solutions (except those that are
purchased as sterile and nuclease-free). Wipe down pipetters and bench with RNase Zap as instructed by
manufacturer.

MATERIALS
Item Description

Company

Cat. No.

Trizol Reagent
Chloroform
Rneasy Mini Kit (50)
Rnase-free Dnase set (50)
Rnase Zap
UltraPure Dnase/Rnase-Free Distilled water
Ethanol (100%) *
1.5 mL micro-centrifuge tubes *
Disposable pestles; 1.5mL, Plastic *
Aerosol Resistant Tips (1000E) *
Aerosol Resistant Tips (200) *
Aerosol Resistant Tips (20) *
Aerosol Resistant Tips (10) *
Eppendorf ® Microcentrifuge 5415 R

Invitrogen
EMD Chemicals
Qiagen
Qiagen
Ambion
Invitrogen
AAPER Alcohol
VWR
VWR
Molecular BioProducts
Molecular BioProducts
Molecular BioProducts
Molecular BioProducts
Eppendorf

15596-025
CX1055-6
74104
79254
AM9780/AM9782
10977-015

* Dnase / Rnase-free grade

101093-206
KT749521-1590
REF2079E
REF2069
REF2149P
REF2139
-------

Unit Size
100 mL
500 mL
50 rxn
50 rxn
250 mL
500 mL
500 mL
500 tubes
100 pestles
100 Tips
96 Tips
96 Tips
96 Tips
-------

¾ Cooled centrifuge is necessary for RNA isolation.5mL Rnase-free micro-centrifuge tubes. ¾ Store Buffer RDD (provided in the Rnase-free Dnase set) at 4 oC.5mL micro-centrifuge tube (Rnase/Dnase-Free. ¾ Recommend that Buffer RDD is added to the Dnase enzyme just prior to use. as well. I process 8-extraction samples at a time and each sample requires 5 μl of the enzyme. o For example. o (i. make aliquots of a desired volume. Thawed enzyme may be store for up to 1 week at 4 oC. Store aliquots for up to 6 months at -20 oC. add 550µl of nuclease-free water (provided in the Rnase-free Dnase set). one 2-mL collection tube. So I make aliquots of 40 μl in 0. and a single micro-centrifuge tube per sample. ¾ IMPORTANT: DNase enzyme is supplied as a lyophilized protein. grade). . On ice.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. DNase enzyme is sensitive to physical denaturation. 3. and two 1. 4. Do not vortex ¾ Before using for the first time. add 4 volumes of 96% 100% ethanol. as indicated on the bottle. Write the date on the bottle. Be sure to check the “YES” box on the cap to indicate that ethanol was added. Cool Centrifuge to 4 oC. Prepare Buffer RPE. to obtain the working solution. ¾ Label a single mini-spin column with collection tube attached. 2. ¾ To avoid repetitive freezing and thawing of the enzyme. Before using for the first time. Dnase enzyme is sensitive to physical denaturation.5mL micro-centrifuge tubes (one supplied in RNeasy Mini Kit and one supplied by user).October 11. a second 2-mL collection tube. (Included in RNeasy Kit) ¾ RPE buffer is supplied as a concentrate. prepare DNase stock solution from the QIAGEN Rnase-free Dnase set.e. Decontaminate work space and equipment with RNase ZAP following manufacturer’s instructions (printed on the bottle). 5. 2007 PROCEDURE Preparations 1. Solution can be stored at room temperature. These materials are provided in the kit and should be handled with Rnase-free gloves to prevent contamination. Setup the following for each sample: one RNeasy mini-spin column. Indiana University. Mix by inverting gently. The user will supply a second 1. 5 μl x 8-extraction samples = 40 μl) ¾ Do not freeze aliquot after thawing.

add 400 µl of TRIZOL Reagent. Mix by inverting 10 times. Assume that a single mature female equals 1 μl in volume. Shake tube vigorously by hand for 15 seconds and incubate for 2 to 3 minutes at 15°C to 30°C. Incubate homogenized tissue in TRIZOL Reagent for 5 minutes at room temperature (15°C to 30°C) to permit the complete dissociation of nucleoprotein complexes. 2007 Cell Lyses and RNA Extraction with Organics FOR LIVE OR FROZEN DAPHNIA TISSUE 1.000 × g for 15 minutes at 2 to 8°C.October 11. 3. ¾ Disruption and homogenization of tissue can be performed by crushing Daphnia with disposable plastic pestle by hand (~5 minutes) or using a battery operated homogenizer. Transfer upper aqueous phase (approx. ¾ For 10 mg – 100 mg or 10 μl – 100 μl of Daphnia. Add one volume of TRIZOL Reagent. 7. 8. Wash down the blue plastic pestle with a second volume of TRIZOL Reagent (equivalent to amount used for step 2 above).8 – 1 mL of TRIZOL Reagent can be stored at -80 oC for up to 1 month. Following centrifugation. the number of individuals is a sufficient to estimate the volume of tissue. add 500 µl of TRIZOL Reagent. 6. weighing the sample using a metric balance or scale is the most accurate method. The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization. Add 200 µl of Chloroform per 1 mL of TRIZOL Reagent. NOTE: Tissue homogenized in 0. The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization. an interphase. and a colorless upper aqueous phase. ¾ Incomplete homogenization will lead to significantly reduced yields and can clog RNeasy column. 5. RNA remains exclusively in the aqueous phase. 2. Determine the amount of tissue.600 rcf for 15 minutes at 4°C ¾ Centrifuge the samples at no more than 12. Using a blue plastic pestle. phenol-chloroform phase. ¾ For FROZEN samples. homogenize tissue sample in the TRIZOL Reagent. 540 μl) to a NEW Rnase-free 1. Save the organic phase if isolation of DNA or protein . Indiana University. ¾ For 1 mg – 10 mg or 1 μl – 10 μl of Daphnia.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. 9.5 mL microcentrifuge tube (supplied by user). 4. the mixture separates into a lower red. Centrifuge at 11. ¾ For LIVE samples.

Transfer precipitated RNA to a Qiagen RNeasy mini-spin column. To continue RNA purification. Discard the flow through. Centrifuge at 9. 79-81. increase the amount of Buffer RW1 to 700 μl. Precipitate RNA by adding 0. ¾ Centrifuge at 9. 50rxn) is a recommended alternative for removal of contaminating DNA from RNA preparations. 11. Proceed to Step 4 of this section. 3. 1. Ambion Dnafree (AM1906. divide the precipitate between two RNeasy mini-spin columns. RNeasy Mini Protocol for RNA Clean-up. loading volume: 700 μl ¾ Max. On-Column Dnase Treatment: If on-column DNase treatment using RNase-Free DNase Set is NOT desired. Discard flow through. Replace column into same 2 mL collection tube. ¾ Max. RNA Purification & On-column DNase treatment with RNeasy Mini Kit Note: The following steps are modified from the protocol outlined in detail in "RNeasy Mini Handbook.600 rcf for 30sec at room temperature (15 – 30 °C).600 rcf for 30sec at room temperature (15 – 30 °C)." p. Centrifuge at 9.asp#mini).Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. ¾ Add to column with 350 µl of Buffer RW1. Discard flow through. Discard flow through after each centrifugation step.com/literature/rnalit. 10. Discard flow through. ¾ Incubate for 15 minutes room temperature (15 – 30 °C). . Indiana University. ¾ Pipet the DNase I incubation mix (40 µl) directly onto the RNeasy silica-gel membrane of the RNeasy mini-spin column. ¾ For samples with expected yields > 100 μg of Total RNA. 2. ¾ Wash column with 350 µl of Buffer RW1.October 11. (http://www. Mix by gently inverting 10 times.600 rcf for 30sec at room temperature (15 – 30 °C). 2007 is desired (See manufacture’s instructions for DNA or protein isolation). Mix by gently pipetting. load aliquots successively onto the RNeasy mini-spin column and repeat centrifugation.qiagen.5x volume of 100% room temperature Ethanol. ¾ If the volume of precipitated RNA exceeds 700µl. Replace mini-spin column into same 2 mL collection tube. ¾ Centrifuge at 9. binding capacity: 100 μg ¾ Exceeding the binding capacity of the mini-spin column (> 100 μg) will significantly reduce yield and quality of the recovered RNA.600 rcf for 30sec at 4°C. ¾ Add 35 µl Buffer RDD to 5 µl DNase I stock solution.

Centrifuge at 9. 10.000 rcf for 2minutes at room temperature (15 – 30 °C). ¾ Buffer RPE is supplied as a concentrate. 8. ¾ It is important to remove trace amount Buffer RPE. Discard flow through.1x volume 5 M NH4OAc ƒ 2.5x volume 100 % Ethanol ƒ Mix by inverting 10 times. o Ethanol Precipitation ƒ 0. Centrifuge at 9.600 rcf for 1 minute at room temperature (15 – 30 °C). 11.5 mL micro-centrifuge tube (supplied in RNeasy Mini Kit). remove the RNeasy silica-gel mini column from the collection tube carefully so the column does not contact the flow-through as this will result in carryover of ethanol. 9. 13.600 rcf for 30sec at room temperature (15 – 30 °C). 7. repeat the elute step (Step 9 & 10) as described with a second volume of RNase-free water. Wash column with 500 µl of Buffer RPE. Carefully remove mini-spin column. 2007 4. Store RNA at -80°C. 5. ¾ If the expected RNA yield is >30 µg. Indiana University. ¾ RNA samples eluted with water can be stored for up to 1 month at -80°C. Discard flow through.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. pipet 30 µl RNase-Free water directly onto silica-gel membrane. . 12.October 11. Centrifuge at 9. Wash column with 500 µl of Buffer RPE. See preparation section at the beginning of this protocol for details. Otherwise this will result in ethanol carry over and reduce the quality of the eluted sample. RNA can be precipitated and stored in ethanol for up to 2 years. Carefully remove mini-spin column. ¾ For long term storage. Incubate for 1 minute at room temperature. Place column in NEW 2 mL collection tube (supplied in RNeasy Mini Kit). Collect and measure each elution separately on Nanodrop. 6. Ensure that ethanol is added to Buffer RPE before use. Replace column into same 2 mL collection tube. ¾ Following the centrifugation of Buffer RPE. To elute. Transfer RNeasy column to a NEW 1. ƒ Store at -80°C. Centrifuge at 16. Carefully remove mini-spin column. Second elution is sometimes less pure.600 rcf for 30sec at room temperature (15 – 30 °C).

A prompt will appear.3 μl of Total RNA sample to pedestal and click Measure. Buff top and bottom pedestal with a Kimwipe. . 11.3 μl of nuclease-free water to pedestal. To quantify sample. ¾ Select “Nucleic Acid”. ¾ Sample type: “RNA – 40” ¾ Extinction coefficient = 40 6. If another elution buffer was used. Click BLANK to calibrate the instrument. ⋅ ⋅ ¾ The concentration of RNA is determined by measuring the absorbance at 260 nm (A260). Lower arm and click OK. Load 1. ¾ If water was used to elute the sample. To BLANK. 2. Launch Nanodrop software to start application and adjust setting for RNA quantification. 9. Buff pedestal both top and bottom of pedestal after every measurement. the reading at 260 (A260) should be greater than 0. Indiana University.3 μl of Elution buffer and click Measure. Instrument is now ready to measure concentration of Total RNA sample. Select “Sample Type” from drop-down menu.3 μl of elution buffer in which RNA is dissolved. Apply pressure gently and stroking 10 times with Kimwipe.October 11.0 If the ratio is lower. that is the buffer used to calibrate the instrument. UltraPure Dnase/Rnase-Free Distilled water Aerosol Resistant Tips (10) * Kimwipes (small) Total RNA (eluted in water) Invitrogen Molecular BioProducts 10977-015 REF2139 Unit Size 500 mL 96 Tips * Dnase / Rnase-free grade 1. Reading should be close to zero.15. To make sure instrument is calibrated properly. apply 1. Be sure to enter a “Sample Name” for each sample and measurement reading. the blank measurement is done with the same water sample used to elute RNA during purification.2 If the ratio is low. No. ¾ “Pure RNA”: 260/230 ratio range of 1. 3. apply 1. apply to the pedestal 1. 10. this may indicate the presence of protein. ¾ “Pure RNA”: 260/280 ratio of ~ 2. 8. 7. 2007 RNA Quantitative Assessment: Determine concentration Item Description Company Cat. 4. 5. this may indicate the presence of co-purified contaminants.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. phenol or other contaminants that absorb at or near 280 nm.8 – 2. To ensure significance.

Gently mixing Total RNA sample by finger-flicking the micro-centrifuge tube prior to measuring the concentration is recommended. ƒ Use a 1.0ul of deionized water to wash pedestal and wipe dry with a kimwipe. visually confirm the water column is completely formed. ƒ RNA sample may not be homogenized. strange results will occur.October 11. 2007 ¾ “Pure RNA”: Smooth curve.0 ul sample size when measuring. Strange results occur when the liquid sample column is not completely formed during the measurement. ƒ If this does not resolve the issue. Use 2. While making a measurement. . Indiana University. Recommend QIAGEN RNA Mini Kit: RNA Cleanup protocol (See Manufacturer’s Manual). ƒ Redo the blank setup. If blank measurement is not done properly. ¾ Trouble shooting: ƒ Make sure sample pedestal is clean. Re-measure the sample.5 – 2. RNA sample may need to be re-purified.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.

¾ Follow manufacturer’s instructions to assess the quality of the Total RNA. Rnase Zap UltraPure Dnase/Rnase-Free Distilled water PCR strip w/ cap (8 tubes/strip) * Aerosol Resistant Tips (1000E) * Aerosol Resistant Tips (200) * Aerosol Resistant Tips (20) * Aerosol Resistant Tips (10) * Eppendorf ® Microcentrifuge 5424 NanoChip Agilent RNA 6000 Nano Kit 0. 2. ¾ Concentrate 400 ng Total RNA to a volume of 1. 2007 Total RNA Qualitative Assessment: Bio Analyzer It is likewise important to guard against sources of dust and nucleases. We recommend using barrier pipette tips. and filtering all solutions (except those that are purchased as sterile and nuclease-free).5 μl in a 0. ƒ Minimal high-molecular weight contamination. Indiana University. Over loading the chip with Total RNA leads to inaccurate assay results. ƒ Low noise between peaks ƒ Minimal low-molecular weight contamination.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.October 11. 3. . ¾ Dilute the sample to a concentration of 300 ng/ μl. High molecular weight contamination may indicate the presence of a contaminant (i.5 μl) to a final concentration of 300 ng/μl. Prepare Total RNA sample (vol = 1. Wipe down all electrophoresis equipment.e. Centrifuge briefly to collect the contents ¾ Keep sample on ice. Set up Bioanalyzer software and adjust settings for Total RNA quantification according to manufacture’s instructions.5 mL Safe Lock micro-centrifuge tubes * Ambion Invitrogen VWR Molecular BioProducts Molecular BioProducts Molecular BioProducts Molecular BioProducts Eppendorf Aligent Agilent AM9780/AM9782 10977-015 20170-004 REF2079E REF2069 REF2149P REF2139 022620401 5067-1511 kit Unit Size 250 mL 500 mL 125 strips 100 Tips 96 Tips 96 Tips 96 Tips ------25 chips 1. DNA). ⋅ High Quality RNA Profile ƒ Distinct 18S and 28S peaks.2 mL micro-centrifuge tube. No. Item Description Company Cat. Mix by gently pipetting. pipettors and bench with RNase Zap as instructed by manufacturer. Interpretation of Bioanalyzer results: ¾ Assess quality of Total RNA from electrophrograms of RNA ribosomal peaks.

ƒ Low-molecular weight contamination may indicate degradation. High molecular weight contamination may indicate the presence of a contaminant (i.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. but peaks are even in height. DNA).e. ⋅ Moderate to Low Quality RNA Profile ƒ 28S peak is considerably lower than 18S peak. ƒ Minimal high-molecular weight contamination. ƒ Abundant low-molecular weight contamination may indicate degradation. High molecular weight contamination may indicate the presence of a contaminant (i. ƒ Low noise between peaks ƒ Minimal low-molecular weight contamination. DNA).e. ƒ Minimal high-molecular weight contamination. DNA). 2007 ⋅ Moderate to High Quality RNA Profile ƒ Distinct 18S and 28S peaks. ⋅ Low Quality RNA Profile ƒ 28S peak is not visible compared to 18S peak. High molecular weight contamination may indicate the presence of a contaminant (i. Indiana University. ƒ Minimal high-molecular weight contamination.e. .October 11.

1 mL of water in the tube. Once all water is removed from sample. ¾ Daphnia can be temporarily stored in this collection tube for up to 5 minutes while continuing with the remainder of the samples collections. Expel air gently to displace any animals obstructing the pipette tip.5 mL micro-centrifuge tube. Remove tube of frozen tissue from liquid nitrogen with forceps.5 mL micro-centrifuge tubes * Pasteur pipet Liquid Nitrogen Long Forceps VWR ---------------- 101093-206 ------------------- Unit Size 500 tubes ------------------- * Dnase / Rnase-free grade 1. ¾ Gently remove water from 1.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.5 mL micro-centrifuge tube. collect adults directly from the culture. No. 2007 Freezing Daphnia: Whole Body Tissue Preparation MATERIALS Item Description Company Cat. collect desired sample in a labeled 1. 1.October 11.5 mL micro-centrifuge tube with Pasteur pipette. 3. 2. Be sure to leave approx. Using a sterile Pasteur pipette. Remove water with Pasteur pipette. Store at -80 oC. Indiana University. . immediately freeze tube of tissue in liquid nitrogen. slowly pour culture into a smaller glass dish or beaker. Freeze Daphnia in liquid nitrogen and store at -80 oC until proceeding to Total RNA isolation and purification. ¾ When collecting from small cultures (beakers). Collect Daphnia sample in 1. Place the tip of a sterile pipette at the bottom of the tube. ¾ When collecting from a large culture (gallon jars).

Pour gel and let solidify. Add 1 μl of Loading dye. ¾ Concentrate 1 μg of RNA to a volume of 1 μl.0500 ------------------------------------- 250 mL 500 mL 500 tubes 125 strips 100 Tips 96 Tips 96 Tips 96 Tips ------100 grams 100 grams 500 mL ------------------------------------- 1. and filtering all solutions (except those that are purchased as sterile and nuclease-free). ¾ Adjust volume with water and allow solution to cool to 55°C.0) * 1 X TBE buffer * Agarose (powder) 6 X Loading Dye * Electrophoresis apparatus Casting tray and comb Ambion Invitrogen VWR VWR Molecular BioProducts Molecular BioProducts Molecular BioProducts Molecular BioProducts Eppendorf Sigma-Aldrich Sigma-Aldrich AppliChem GmbH ------------------------------- AM9780/AM9782 10977-015 101093-206 20170-004 REF2079E REF2069 REF2149P REF2139 ------G3272 E7637-1G A0937. Air bubbles will interfere with the nucleic acid separate and lead to poor results. pipettors and bench with RNase Zap as instructed by manufacturer. 2. Wipe down all electrophoresis equipment.5% w/v gel. To 1 μl concentrated Total RNA sample. 3. We recommend using barrier pipette tips.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.5% w/v Agarose gel with 1X TBE with 20 mM Guanidine-HCl.5% w/v gel. ¾ Add 0. 5. ¾ For a 50 mL volume 1. Unit Size Item Description Company Cat. Push any air bubbles using a sterile tooth pick to be bottom of the gel. No. ¾ ¾ ¾ ¾ add 4 μl Formamide-TE buffer Denature by heating at 70°C for 2 minutes Briefly centrifuge to collect sample. Prepare a 1. Add Guanidine-HCl to a final concentration of 20mM.October 11.75g Agarose to 50 mL of 1X TBE buffer for a 50 mL volume 1. ¾ Pour gel solution into casting tray. Add 2 μl Ethidium Bromide (10mg/mL) to cool Agarose solution. . Indiana University. Rnase Zap UltraPure Dnase/Rnase-Free Distilled water 1. 4. Prepare 1 μg Total RNA sample for electrophoresis. 2007 Total RNA Qualitative Assessment: Protocol for RNA Gel with Guanidine-HCl It is likewise important to guard against sources of dust and nucleases.5 mL micro-centrifuge tubes * PCR strip w/ cap (8 tubes/strip) * Aerosol Resistant Tips (1000E) * Aerosol Resistant Tips (200) * Aerosol Resistant Tips (20) * Aerosol Resistant Tips (10) * Eppendorf ® Microcentrifuge 5415 R Guanidine hydrochloride (powder) Ethdium Bromide (powder) Formamide 1X TE buffer (pH 7. ¾ Ethidium Bromide is a carcinogen and should be handled according to safety protocols. ¾ Boil to dissolve Agarose. 6. add 1 mL of 1M guanidine-HCl.

¾ Remove comb. 2007 7.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC. Prepare gel for electrophoresis. Visualize RNA ribosomal bands with UV shadowing. Electrophoresis for 1. High-molecular weight contamination may indicate DNA contamination. 8. 11.October 11. . and submerge gel into electrophoresis chamber filled with electrophoresis buffer. 9. Load one sample per gel into the wells. 12. Little smearing between bands. 10. Indiana University. The high molecular weight band should be about twice as intense as the low molecular weight band. Low-molecular weight contamination may indicate RNA degradation.0 hours at 97 mV.

1M Tris-HCl. distilled deionized water.77 g Guanidine-HCL in 50 mL of filtered. 6 X Gel loading buffer with glycerol recipe 25.0 with 6 N NaOH. 1X TE To 990 mL deionized. Use a dark glass bottle. distilled nuclease free water. Then bring volume to 500 mL and sterilize by autoclaving. pH 8.0 g Ethidium bromide in 100 mL filtered.05 g of Na2EDTA in 400 mL deionized.0): 500 mL Dissolve 95.0 g Boric Acid 7. 2007 Solutions Formamide-TE buffer Add 300 μl formamide to 100 μl 1x TE 1M Guanidine-HCL Dissolve 4.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.14) in 100mL H2O (adjust pH with concentrated HCl) .0 12.5 M EDTA (pH 8.0 mg Bromophenol blue 25. add 1. 1x TBE electrophoresis buffer recipe (working solution) To 700 mL of filtered.0 g Tris Base 55. add 54. 10 mg/mL Ethidium bromide recipe Dissolve 1. Autoclave to sterilize.October 11.0 mL glycerol.4 g EDTA (disodium salt) Stir to dissolve.0 g NaOH 108. Add 10mL 1M Tris-HCl (pH 8. if possible.0 mg Xylene cyanol FF 3.0 mL 0. distilled nuclease free water.0) Add 200µl 0. 20 minute sterilization. distilled deionized water. distilled deionized water When dissolved.0 g Tris base 27. Autoclave to sterilize.5 g Boric acid 20.0) Bring to a final volume of 1 L. distilled deionized water. Bring to a volume of 10 mL with distilled water.5 M EDTA 5 M EDTA (pH 8. Adjust pH to 8. wrap container in aluminum foil and keep in the dark. Indiana University.1g Tris base (MW 121. 10 X TBE electrophoresis buffer recipe (Stock Solution) To 700 mL of filtered. Bring up final volume of 1 L. Store at 4°C. 20 minute exhauste.

Indiana University.Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy Mini Kit ©DGC.October 11. 2007 .