Sean Lauber:Reverse Transcription using Superscript II: Difference between revisions
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Using superscript II (Invitrogen CAT# 18064-022) | Using superscript II (Invitrogen CAT# 18064-022) | ||
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1. Put appropriate amount of RNA (eg. 180 ng) into a tube | 1. Put appropriate amount of RNA (eg. 180 ng) into a tube | ||
2. Bring the final volume up to 10 μL. <b> If your solution is too dilute you can use a vacuum centrifuge to evaporate excess volume and then bring it up to 10 μL</b> | 2. Bring the final volume up to 10 μL. <b> If your solution is too dilute you can use a vacuum centrifuge to evaporate excess volume and then bring it up to 10 μL</b> | ||
10 μL RNA | 10 μL RNA | ||
1 μL dNTPs | 1 μL dNTPs | ||
1 μL 0.2 μg/μL 6-mer (this acts as the primer for initiating cDNA synthesis) | 1 μL 0.2 μg/μL 6-mer (this acts as the primer for initiating cDNA synthesis) | ||
3. Heat at 65*C for 5 min (use the thermocycler, program "SS2RT") | 3. Heat at 65*C for 5 min (use the thermocycler, program "SS2RT") | ||
4. Quickly chill the samples on ice | 4. Quickly chill the samples on ice | ||
5. Add: | 5. Add: | ||
4 μL 5x First-strand buffer | 4 μL 5x First-strand buffer | ||
2 μL 0.1 M DTT | 2 μL 0.1 M DTT | ||
6. Incubate at 25*C for 2 min | 6. Incubate at 25*C for 2 min | ||
7. Add: | 7. Add: | ||
1.5 μL DEPC-treated water | 1.5 μL DEPC-treated water | ||
0.5 μL SSII | 0.5 μL SSII | ||
8. Incubate at 25*C for 10 min | 8. Incubate at 25*C for 10 min | ||
9. Incubate at 42*C for 50 min | 9. Incubate at 42*C for 50 min | ||
10. Incubate at 70*C for 15 min to inactivate the enzyme | 10. Incubate at 70*C for 15 min to inactivate the enzyme | ||
11. Store at -20*C or at 4*C overnight | 11. Store at -20*C or at 4*C overnight | ||
Revision as of 07:56, 30 March 2012
Using superscript II (Invitrogen CAT# 18064-022)
First you need to determine how much cDNA you want to generate. How much will you need for TaqMan? I generally use 10 ng/Taqman reaction and this is done in triplicate. Also considering the number of probes, you will need:
10 ng * 3 * # of probes = ng of cDNA required
So if I wanted to look at 6 different probes (including the housekeeping gene), I would need 180 ng of cDNA so I will need 180 ng of RNA (assuming 1:1 conversion). To control for potential differences in efficiency of cDNA synthesis, you must use the same amount of cDNA for each reaction. So you will need to provide 180 ng of RNA for each reverse transcription reaction. So if you have 4 samples, you need to figure out what volume you will need to give you 180 ng of RNA.
1. Put appropriate amount of RNA (eg. 180 ng) into a tube
2. Bring the final volume up to 10 μL. If your solution is too dilute you can use a vacuum centrifuge to evaporate excess volume and then bring it up to 10 μL
10 μL RNA 1 μL dNTPs 1 μL 0.2 μg/μL 6-mer (this acts as the primer for initiating cDNA synthesis)
3. Heat at 65*C for 5 min (use the thermocycler, program "SS2RT")
4. Quickly chill the samples on ice
5. Add:
4 μL 5x First-strand buffer 2 μL 0.1 M DTT
6. Incubate at 25*C for 2 min
7. Add:
1.5 μL DEPC-treated water 0.5 μL SSII
8. Incubate at 25*C for 10 min
9. Incubate at 42*C for 50 min
10. Incubate at 70*C for 15 min to inactivate the enzyme
11. Store at -20*C or at 4*C overnight
