RevertAid？ First strand cDNA synthesis kit
(#K 162 1 10 times)
- 1
Analysis license
warn
It is suggested that the synthesis of the first-strand cDNA should be carried out under the condition of eliminating RNase pollution. Pipette tips and test tubes must be treated with 0. 1% DEPC (soaked in 0. 1% water solution overnight, heated at 100℃ for 30 minutes and autoclaved). Gloves are highly recommended.
Important!
Store at -20℃.
(If the control RNA is stored at -70℃ for a long time).
The control RNA avoids repeated freezing and thawing. The control RNA was melted and operated on ice.
A lot. : 12 10
See the package label for the shelf life.
- 2
The kit is designed to prepare full-length first-strand cDNA from RNA template. RevertAid？ The first-strand cDNA synthesis kit relies on a genetic engineering product: Moloney mouse leukemia virus reverse transcriptase (RevertAid? M-MuLV RT). This allows the synthesis of full-length cDNA from a long template (maximum 13 kb). RevertAid？ The position of the first-strand cDNA synthesized by M-MuLV RT is determined by different primers:
Random hexamer primer: All RNA in the total RNA is a template for cDNA synthesis in non-specific positions on the RNA template.
Oligo(dT) 18: at the 3'- end of poly(A)+ mRNA. In this case, only mRNA with 3'- polyadenylate tail is the template for cDNA synthesis.
Sequence-specific primer: at the primer binding position.
The first-strand cDNA synthesized by this system can be used as PCR* template. Because the reaction conditions of cDNA synthesis and PCR are the same, the cDNA reaction mixture can be directly added to the PCR mixture. The synthesized first-strand cDNA can also be used as a template for the second-strand synthesis. Radiolabeled DNA can be used as a hybridization probe.
1. 1 kb RNA with 3'- polyadenylate tail was used as control.
* The *PCR process is undertaken by Hoffmann-laroche.
- 3
Package content
1.RevertAid？ M-MuLV reverse transcriptase (200u*/? l):
15? L enzyme is dissolved in storage buffer: 50 mm tris-HCl (pH 8.3), 0. 1 m NaCl, 1 mm EDTA, 5 mm DTT, 0. 1% Triton? X- 100 and 50% glycerol.
2. RNA? Ribonuclease inhibitor (20u**/? l):
15? Dissolve the enzyme in storage buffer: 20 mm hepes-NaOH (ph 7.5), 50 mm NaCl, 8 mm dtt, 0.5 mm eluent? Detergent and 50% glycerin.
3.5x reaction buffer:
100? 15x reaction buffer: 250 mm tris-HCl (pH 8.3 at 25℃), 250 mm KCl, 20 mm MgCl2, 50 mm DTT.
4 4. 10/0mm dNTP mixture:
30? L 10mM dGTP, dATP, dTTP, dCTP aqueous solution.
5. oligonucleotide (dT) 18 primer:
15? l 0.5？ g/？ L (15A260 units/ml) aqueous solution.
6. Random hexamer primer:
15? l 0.2？ g/？ L (6A260 units/ml) aqueous solution.
7. Control primer:
15? l 10pmol/？ L( 1.7a 260 unit /ml) 17 polymer aqueous solution.
8. Control RNA:
15? L 1. 1 kb 65438 with 3'-poly(A) tail +0. 1 kb RNA, 0.5? g/？ l .
9.DEPC treated water:
2 x 1.5 ml deionized water on amilili-q? Deionization and DEPC treatment in the plant.
* A unit RevertAid? M-mulvrt: 1 nanomole dTMP binds to the polynucleotide fragment (adsorbed in DE-8 1) at 37℃ 10 min.
* * One unit of RNA? RNase inhibitor: Inhibition of 5 ng RNase A activity by 50%.
- 4
operation sequence/order
1. Synthesize the first cDNA chain suitable for PCR amplification.
① Prepare the following reaction mixture in a test tube on ice:
Template RNA*:
Total RNA 0. 1-5 μ g.
Or poly (a)+RNA10ng-0.5μ g.
Or specific RNA 0.01pg-0.5 μ g.
Primer:
Oligonucleotide (dT) 18 primer (0.5 μ g/L) 1 L.
Or random hexamer primer (0.2 microgram/microliter) 1 microliter.
Or sequence-specific primer15–20 pmol.
DEPC treated water to 12 microliter.
Gently mix and centrifuge for 3-5 seconds.
② Incubate the mixture at 70°C for 5 minutes, cool it with ice, centrifuge briefly, and collect the precipitate.
③ Place the test tube on ice and add the following components in the specified order:
5 times the reaction buffer 4 microliters
Ribonucleic acid? Ribonuclease inhibitor (20u/μl) 1μl
10mm dNTP mixture 2 microliters.
Mix gently, centrifuge briefly, and collect precipitate.
④ Incubate at 37℃ for 5 minutes (random hexamer primer at 25℃ for 5 minutes).
* The amount of total RNA or poly(A)+ RNA required for the reaction depends on the expression level of the gene.
- 5
⑤ Join RevertAid? M-MuLV reverse transcriptase (200 u/μ l) 1 μ l
The final volume was 20 microliters.
⑥ Incubate the mixture at 42 C for 60min (random hexamer primer at 25°C10min, and finally at 42 C for 60min).
⑦ Heat at 70℃ for 65438 00 minutes to terminate the reaction. It's cold and cold.
The synthesized first-strand cDNA can be directly used for PCR amplification. PCR can be completed with the following products:
Taq DNA polymerase (recommended) (# ep040 1, # ep0402, # ep0403, # ep0404);
Taq DNA polymerase (natural without BSA) (# ep028 1, # ep0282, # ep0283, # ep0284);
Taq DNA polymerase (natural, without BSA) (# ep007 1, # ep0072);
2mM dNTP mixture (# r024 1, # r0242);
10mm dNTP mixture (# r0 19 1, # r0 192).
note:
1. poly(A)+ RNA does not need to be isolated from the total RNA first, but the yield and purity of the final product can be improved.
2.RNA samples should not be contaminated by genomic DNA.
3.oligo(dT) 18 primer does not need to be optimized, but according to the average length of cDNA synthesized in the reaction, the ratio of random hexamer primer to RNA is strict. Increasing the ratio of hexamer /RNA will lead to higher yield of shorter (~500 bp) cDNA, while decreasing the ratio will lead to longer product.
4. Increasing the reaction temperature to 45°C can alleviate the secondary structure problem rich in GC mRNA.
5. Analysis of reaction products (see Chapter 4) cDNA needs [32P] radioactive labeling. Join RevertAid for this? Add 10μCi[? -32P]dNTP (such as dATP) is added to the reaction mixture. Add 1μl 0.5M EDTA to terminate the reaction and put it on ice.
6. The synthesized cDNA should be stored at -20℃ ..
- 6
2. Synthesize the first-strand cDNA suitable for the second-strand synthesis.
① Prepare the following reaction mixture in a test tube on ice:
Template RNA*:
Polyadenylic acid+RNA 1 μ g
Or specific RNA 0.5-1μ g.
Primer:
Oligonucleotide (dT) 18 primer (0.5 μ g/L) 1 L.
Or random hexamer primer (0.2 microgram/microliter) 1 microliter.
Or sequence-specific primer 100pmol.
DEPC treated water to 12 microliter.
Gently mix and centrifuge for 3-5 seconds.
② Incubate the mixture at 70°C for 5 minutes, cool it with ice, centrifuge briefly, and collect the precipitate.
③ Place the test tube on ice and add the following components in the specified order:
5 times the reaction buffer 4 microliters
Ribonucleic acid? Ribonuclease inhibitor (20u/μl) 1μl
10mm dNTP mixture 2 microliters.
Mix gently, centrifuge briefly, and collect precipitate.
④ Incubate at 37℃ for 5 minutes (random hexamer primer at 25℃ for 5 minutes).
⑤ Join RevertAid? M-MuLV reverse transcriptase (200 u/μ l) 1 μ l
The final volume was 20 microliters.
- 7
⑥ Incubate the mixture at 42 C for 60min (random hexamer primer at 25°C10min, and finally at 42 C for 60min).
⑦ Heat at 70℃ for 65438 00 minutes to terminate the reaction. It's cold and cold.
The synthesized first-strand cDNA can be used for the second-strand synthesis. The following products can be used to complete the synthesis:
DNA polymerase I (#EP004 1, # ep0042);
T4 DNA ligase (# el00 14, # el001,# el0012);
Ribonuclease H (#EN020 1, # en0202);
Nuclease S 1 (#EN032 1).
notes
1. In order to improve the specificity and efficiency of synthesis, the poly(A)+ fragment must be separated from the total cell RNA.
2.oligo(dT) 18 primer does not need optimization conditions, but according to the average length of cDNA synthesized in the reaction, the ratio of random hexamer primer to RNA is strict. Increasing the ratio of hexamer /RNA will lead to higher yield of shorter (~500 bp) cDNA, while decreasing the ratio will lead to longer product.
3. Increasing the reaction temperature to 45°C can alleviate the secondary structure problem rich in GC mRNA.
4. Analysis of reaction products (see Chapter 4) cDNA needs [32P] radioactive labeling. Join RevertAid for this? Add 10μCi[? -32P]dNTP (such as dATP) is added to the reaction mixture. Add 1μl 0.5M EDTA to terminate the reaction and put it on ice.
5. The synthesized cDNA should be stored at -20℃.
- 8
3. Synthesize the first chain with high specific radioactivity.
① Prepare the following reaction mixture in a test tube on ice:
Template RNA*:
Polyadenylate+RNA 0.5 μ g
Primer:
Oligonucleotide (dT) 18 primer (0.5 μ g/L) 1 L.
Or random hexamer primer (0.2 μ g/microliter) 1.5 microliter.
Or sequence-specific primer 100pmol.
DEPC treated water to 8 μ l.
Gently mix and centrifuge for 3-5 seconds.
② Incubate the mixture at 70°C for 5 minutes, cool it with ice, centrifuge briefly, and collect the precipitate.
③ Place the test tube on ice and add the following components in the specified order:
5 times the reaction buffer 4 microliters
Ribonucleic acid? Ribonuclease inhibitor (20u/μl) 1μl
1 0mm dGTP, dCTP, dTTP mixture1microliter
0. 1 mm dATP* 4 microliter
Mix gently, centrifuge briefly, and collect precipitate.
④ Incubate at 37℃ for 5 minutes (random hexamer primer at 25℃ for 5 minutes).
⑤ Join:
[? -32P]dATP( 10 nanogram/ml) 1 microliter.
RevertAid？ M-MuLV reverse transcriptase (200u/μl) 1μl
The final volume was 20 microliters.
* A separate dNTP solution is not included in this package. Use dNTP set (#R0 18 1) for synthesis. .
- 9
⑥ Incubate the mixture at 42 C for 60min (random hexamer primer at 25°C10min, and finally at 42 C for 60min).
⑦ Add 5 μ l of 0.5m EDTA to terminate the reaction.
⑧ If necessary, add equal volume (25 μ l) of 0.6N NaOH to hydrolyze RNA, and incubate at 70°C for 30 minutes.
Pet-name ruby in Sephadex? Unbound dNTPs was removed by G-50 column chromatography.
The first-strand cDNA with high specific activity (>: 107 dpm/μg) can be used as Southern hybridization probe.
note:
In order to obtain cDNA with higher specific activity (more than 108dpm/μg), the [? -32P]dATP was added to the reaction mixture. If the final reaction system is greater than 20μl, 10μl [? -32P]dATP (10mCi/ml) and transfer the prepared reaction mixture to the test tube (5 steps). Then continue to synthesize.
4. Analyze the first strand cDNA product.
Alkaline agarose gel electrophoresis can only identify or analyze radiolabeled cDNA products.
Determine the yield of the first cDNA chain.
① Spot 1μl sample on two pieces of DE-8 1 filter membrane (1.5x 1.5cm).
(2) Bake the lamp to dry the filter membrane. Keep a filter membrane and use it directly to determine the total radioactivity of the sample.
③ The other filter membrane was washed for 5 minutes10ml 7.5% (w/v) na2hpo4x12h2o for 5 minutes three times; Rinse with water, acetone or 96% ethanol.
- 10
(4) baking lamp to dry the cleaned filter membrane.
⑤ Transfer two filter membranes (unwashed and washed) to a radioactivity counter and count them.
⑥ Calculate the yield of the first cDNA strand with the following formula:
If the final concentration of dNTP is 1.0mM, the number of moles of dATP in (20μl) is 2x 10-8 moles. The number of marked dATP is quite small and can be ignored. However, in the case of cDNA with high specific activity (Chapter 3), the final concentration of unlabeled dATP is 0.02 mm ... In order to calculate the molar dATP in measurement, it is necessary to add the unlabeled molar and the labeled molar.
MW is the average molecular weight of nucleic acid, which is equal to 333x 106 μg/mol. Because all four dNTP participate in the reaction, MW is multiplied by 4.
- 1 1
For example, for example:
If the washed filter membrane produces 4x 104dpm, the unwashed filter membrane produces1.8x106 DPM;
So:
CDNA yield (μ g) = 4x104 dpmx (2x10-8 mol datp) x4x (333x106 μ g/mol) = 0.59 μ g.
1.8x 106dpm
CDNA yield% = 0.59 μ g x 100% = 59%
1 μ g
Alkaline agarose gel electrophoresis analysis
The synthetic products of the first-strand cDNA labeled with [32P] can be analyzed by alkaline agarose gel electrophoresis. Similarly, both synthetic products and DNA markers used should be labeled with [32P].
① Prepare 1.4% agarose gel (30mM NaCl, 2mM EDTA) and balance at least 1h in alkaline electrophoresis buffer (30mM NaOH, 2mM EDTA).
② The sample (1x 105 dpm) was transferred to a separate test tube and diluted with 5μl of water. Add 5μl 2x loading buffer.
(60mM NaOH，2mM EDTA，6% Ficoll？ 400, 0.05% bromophenol blue). The labeled DNA markers should be diluted with the same 2x loading buffer.
Note that 2x loading buffer should be kept at-20 C, or dye should be added before use.
(3) Add the sample and conduct electrophoresis in alkaline agarose gel at 5V/cm until the dye migrates to the position close to 2/3 of the gel.
- 12
④ The gel was soaked in 7% trichloroacetic acid (TCA) for 30 minutes at room temperature.
⑤ The gel is dried in vacuum on a gel dryer or under a multi-layer paper towel pressed by a glass plate for 1-2 hours.
⑥ Cover the dried gel with a plastic cover and expose the X-ray film at room temperature overnight.
Control synthesis
1. 1 kb RNA with 3'- polyadenylate tail was provided as a control.
① Prepare the following reaction mixture in a test tube on ice:
Template RNA*:
Control RNA 0.5 μ g
Primer:
Oligonucleotide (dT) 18 primer (0.5 μ g/L) 1 L.
Or random hexamer primer (0.2 microgram/microliter) 1 microliter.
Or control (sequence-specific) primer (10pmol) 2 μ l.
DEPC treated water to11μ l.
Gently mix and centrifuge for 3-5 seconds.
② Incubate the mixture at 70°C for 5 minutes, cool it with ice, centrifuge briefly, and collect the precipitate.
③ Place the test tube on ice and add the following components in the specified order:
5 times the reaction buffer 4 microliters
10mm dNTP mixture 2 microliters.
Ribonucleic acid? Ribonuclease inhibitor (20u/μl) 1μl
Mix gently, centrifuge briefly, and collect precipitate.
④ Incubate at 37℃ for 5 minutes (random hexamer primer at 25℃ for 5 minutes).
⑤ Join:
[? -32P]dATP( 10 nanogram/ml) 1 microliter.
RevertAid？ M-MuLV reverse transcriptase (200u/μl) 1μl
The final volume was 20 microliters.
- 13
⑥ Incubate the mixture at 42 C for 60min (random hexamer primer at 25°C10min, and finally at 42 C for 60min).
⑦ Add 1μl 0.5M EDTA to terminate the reaction and put it on ice.
⑧ Analysis products (see Chapter 4).
note:
1. When the control RNA is used, the yield of the first-strand cDNA usually exceeds 50%.
2. If the control is synthesized with oligo (dT) 18 or control (sequence-specific) primers, a clear band of 1. 1 kb can be observed. If random hexamer primers are used, several short bands can usually be observed.
- 14
FREQUENTLY ASKED QUESTIONS
Low yield and undetectable products are two obvious signs of reverse transcriptase reaction failure.
Possible causes inspection and remedy
DNA template degradation. Glyoxal and formaldehyde gel electrophoresis were used to detect the integrity of RNA template. The control RNA should have a bright band of 1. 1kb in the gel. When handling sample RNA, be careful not to be contaminated by RNase. The template RNA and control RNA were stored at-70 C to avoid repeated freezing and thawing of samples, and then put on ice after melting.
Ribonuclease contaminated the reaction mixture. Use a control and analyze the resulting product (see synthesis of control). Prepare the reaction mixture under aseptic conditions, always wear gloves, and handle all instruments that come into contact with the sample with DEPC. Mainly does not pollute the solution.
RevertAid？ M-MuLV reverse transcriptase inhibitors (SDS, EDTA, guanidine salt, phosphate, pyrophosphate, polyamine, spermine, spermidine). 1μg control RNA was mixed into RNA samples and synthesized at the same time. If oligo(dT) 18 or control primer is used, the yield of synthesis should be above 50%, and a bright additional band of 1. 1kb can be seen. The RNA sample was precipitated with 96% ethanol and washed with 75% ethanol (DEPC-treated water should be used to prepare ethanol solution). Do not add inhibitors to the reaction mixture.
Incorrect primers are used to analyze products together with other primers. The sequence-specific primers should be complementary to the 3' end of RNA. If the RNA template contains transcription interruption, it is synthesized using random hexamer primers.
The synthesized first-strand cDNA sequence is wrong. Repeated synthesis of cDNA.
- 16
quality management
All reagents in the kit were detected by using polyadenylated RNA transcripts as control templates (specific control primers, oligo(dT) 18, random hexamer primers) for the first-strand cDNA synthesis reaction. .
Quality verification: Birute Gagiliene