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ABOUT US
Applied Biotechnology is manufacturing company for
molecular biology reagents. Also, Applied Biotechnology
company offers many molecular services to the scientific
community. The company mission is offering good quality
molecular reagents and services at reasonable prices to help
the researchers. The company operates by well trained
researcher. Also, we try to offer professional customer
support to our customers. The company has well equipped
molecular diagnostic laboratory. The company headquater is
located in Ismailia province, Egypt.
Molecular
research
services
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9-10
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Electrophoresis
100 bp DNA ladder
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DNA extraction
RNA extraction
Polymerase chain reaction
Gel electrophoresis
Real time PCR
Bacterial and fungal identification
Gene Sequencing
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Storage
RT
-20C
RT
RT
RT
RT
50 preps
12 ml
1 ml
10.5 ml
6 ml
50
12ml
Notes:
1. Please add absolute ethanol to WB1 and WB2 before first use. Mix well and mark the check box
labeled on the bottles to indicate that the ethanol has been added.
2. Please ensure the bottles tightly capped when not in use, prevent reagents from evaporating,
oxidation and pH change.
II. Principles:
The kit has a unique lysis buffer and Protease K to rapidly lyse cell and inactivate cellular nuclease,
then the DNA selectively adsorbs to silicified membrane in high salt solution. Cellular metabolite,
proteins and inhibitors are removed by serial washing by potent two washing buffers. Finally the
purified DNA eluted from silica membrane by low salt elution buffer.
III. Features
1. No need of harmful phenol and chloroform.
2. Simple and rapid. One preparation can be completed in 20 min.
3. Multi-elution ensures high-purified DNA. The DNA yield achieving 3-6g in 100 ul.
V. Procedures
1. Add 200 ul of DNA lysis buffer and 19 ul of proteinase K to the prepared samples in 1.5 ml
centrifuge tubes.
2. Mix vigorously by pipetting and vortex. Please, avoid any splash into the automatic pipette or
leakage from 1.5 ml centrifuge tubes to avoid cross contamination.
3. incubate at 65C for 20 minutes in blood and bacteria. The incubation period should increase
up to 3 hours in case of tissues to ensure proper lysis. N.B the length of incubation varies, if you
find that tissue samples are well homogenate and completely lysed so you can proceed.
4. Vigorous vortexing during incubation to ensure proper lysis.
5. In case of tissue samples or any other samples that is not completely lysis, spin the tube at
12000 rpm for 1 minutes, transfer the supernant into fresh 1.5 ml centrifuge tube. This step is
important to avoid clogging of the spin columns.
6. Add 100 ul of isopropanol or absolute ethanol to the lysate. Mix well by inversion for 4 times.
7. Transfer the lysate into Spin column. Then centrifuge at 12000 rpm for a minutes. Discard
the filterate in the collection tube. If the column clogged by climbs or any debris, please try to
remove the blocking materials from column by tips of automatic pipette and spin the column
again.
8. Add 300 ul of WB1 into the spin column. Spin at 12000 rpm for 30 seconds. Discard the
filterate.
9. Add 300 ul of WB2 into the spin column. Spin at 12000 rpm for 30 seconds. Discard the
filterate.
10. Spin the empty column at 12000 rpm for 1 minutes to get rid of any remaining washing
buffer.
11. Dicard the collection tube and transfer the spin columns into a new 1.5 ml centrifuge
tubes.
12. Add 50 ul of elution buffer into the center of the spin column. Incubate at room
temperature for 5 minutes. Centrifuge at 12000 rpm for 30 seconds.
13. Add another 50 ul of elution buffer into the spin column and repeat the step before.
14. Store the total 100 ul of highly pure DNA in 20C for further applications.
Storage
50 preps
4
RT
37 ml
15 ml
30 ml
20 ml
50
15 ml
RT
RT
RT
RT
Notes:
1 Please add ration ethanol to Buffer RW and 70% ethanol before first use. Mix well and
mark the check box labeled on the bottles to indicate that the ethanol has been added.
2 Please ensure the bottles tightly capped when not in use, prevent reagents from
evaporating, oxidation and pH change.
II. Principles:
The kit has a unique RNA lysis buffer that rapidly lyse cell and inactivate cellular nuclease, then the
RNA selectively adsorbs to silicified membrane. Cellular metabolite, proteins and inhibitors are
removed by serial washing by potent washing buffers. Finally the purified RNA eluted from silica
membrane by low salt elution buffer.
III. Procedures:
Note: Add absolute ethanol to Buffer WB and 70% ethanol.
1 Homogenization:
a. Tissues: Please homogenize tissue in 700 ul of RNA lysis buffer.
b. Blood: Please add 200 ul of blood to 700 ul of RNA lysis buffer, mix well by pipetting.
Vortex vigorously.
C. Fecal matter: Suspend the fecal materials in saline, leave the sample for 5 minutes at
room temperature. Transfer 200 ul of supernatant into 1.5 ml tubes. Add 700 ul of RNA lysis
buffer. Mix well by pipetting. Vortex vigorously.
2 Incubate the homogenized samples for 5 minutes at room temeprature to permit the
complete dissociation of nucleoprotein complexes.
3 Add 0.2 ml of chloroform. Cap sample tubes securely. Vortex vigorously. Incubate them at
room temperature for 2 to 3 minutes.
4 Centrifuge at 12,000rpm for 10 minutes at room temperature.
5 Transfer the aqueous phase to a fresh tube. Add 700 ul of 70% ethanol. Mix by inversion
for 3 times. Transfer the mixture and precipitate to a Spin-column AC (placed in collection
tube). If the mixture is too much, apply the mixture in successive application to the same
Spin-column AC.
6 Centrifuge at 10,000 rpm for 30s at room temperature. Discard the flow through. Reuse
the Spin-column and the collection tube.
7 Add 500l inhibitor removal buffer to the center of Spin-column AC to remove the protein.
Centrifuge at 12,000 rpm for 45s. Discard the flow through.
8 Add 500l Buffer RW. Centrifuge at 12,000rpm for 30s. Discard the flow through.
9 Add 500l Buffer RW. Centrifuge at 12,000rpm for 30s. Discard the flow through.
10 Replace Spin-column AC to the collection tube and spin for 2min to remove the residual
fluid.
11 Place Spin-column AC to a 1.5ml RNase-free centrifuge tube. Apply 50l of elution buffer
to the center of the column. Place it at room temperature for 2min. Centrifuge at
12,000rpm for 1min. return the column to centrifuge tube and add additional 50 ul
elution buffer, spin again. You will have a total of 100 ul RNA.
12 Store the highly purified RNA at -80C until use.
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Amount
1.25 ml
25L reaction
volume
12.5 L
X L
1L
1L
Up to 25L
50 L reaction
volume
25 L
X L
1L
1L
Up to 50L
Step
1
1
2
3
Temperature
95
95
50-60
72
Time
2-5min
30s
30s
1min/1kb
72
5 min
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Amount
1.25 ml
25L reaction
volume
12.5 L
X L
1.5 L
1.5 L
0.5 L
Up to 25L
Step
1
1
2
3
Temperature
95
95
50-60
72
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Time
3 min
15 seconds
30 seconds
30 seconds
2X SYBR Mix
Component
Product
2X Taqman Master Mix
Amount
1.25 ml
Description:
2X SYBR Mix is a 2X concentrate of premix containing DNA polymerase, SYBR Green I, dNTP,
optimized buffer and stabilizer specially designed for real-time PCR with intercalator method.
This mix do not contain any Rox reference dye.
Reaction Setup
Component
25L reaction
volume
12.5 L
X L
1.5 L
1.5 L
Up to 25L
2MasterMix
Template DNA
Upstream Primer (10M)
Downstream Primer (10M)
Sterile Water
Thermal cycler condition
Cycle
1
45
Step
1
1
2
3
Temperature
95
95
50-60
72
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Time
3 min
15 seconds
30 seconds
30 seconds
Product
M-MLV Reverse
Transcriptase200U/l
5First-strand Buffer
5.5 l
22 l
RNase-free H2O
750 l
60 l
15 l
Procedure
1Add the following components:
Template RNA
Primer
0.2-2g
Reverse primer
20 picomole
Or oligo (dt)18
1 ul
Or Random primer
1 ul
Nuclease free water
Up to
13.5 ul
2. If the RNA template is GC-rich or contains secondary structures, incubate at 65C for 5
min. Chill on ice, spin down and place the vial back on ice.
3. Add the following components into each sample:
5First-strand Buffer
4l
0.5 l
2 ul
3. For oligo(dT)18 or gene-specific primed cDNA synthesis, incubate for 60 min at 42C. For
random hexamer primed synthesis, incubate for 5 min at 25C followed by 60 min at 42C.
Note. For GC-rich RNA templates the reaction temperature can be increased up to 45C.
4. Terminate the reaction by heating at 70C for 5 min. The reverse transcription reaction
product can be directly used in PCR applications or stored at -20C.
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Electrophoresis
100bp DNA Ladder
Contains 11 discrete DNA fragments ranging in size from 100bp to 1500bp. This marker is suitable
for sizing linear double-stranded DNA fragments. All bands (except 500bp) are supplied at
approximately 40-50 ng/5l. The 500bp are 100ng/5l as a reference band. This ladder is premixed with loading dye and is ready to use.
Concentration
Reference Band 100 ng/5l, Other Bands 40-50 ng/5l
Storage
The 100 bp ladder is stable for at least 3 months at 4C or room temperature. For long term
storage, store at -20C.
Recommended Loading
3-5 l/Lane.
Contentsbp
100, 200, 300, 400, 500, 600,700, 800, 900, 1,000, 1,500bp
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The 100bp Plus DNA Ladder contains 14 discrete DNA fragments ranging in size from 100bp to
5kb (100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 5000bp). All bands
(except 500bp) are supplied at approximately 40-50 ng/5l. The 500bp are 100ng/5l as a
reference band. This ladder is pre-mixed with loading dye and is ready to use.
Concentration
Reference Band 100 ng/5l, Other Bands 40-50 ng/5l
Storage
The 100 bp ladder is stable for at least 3 months at 4C or room temperature. For long term
storage, store at -20C.
Recommended Loading
3-5 l/Lane.
Contentsbp
100, 200, 300, 400, 500, 600,700, 800, 900, 1,000, 1,500bp
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