cDNA synthesis using superscript II
by A. Untergasser (contact address and download at
www.untergasser.de/lab)
Version: 1.0 - Print
Version (.PDF)
ATTENTION: This is expensive.
One reaction as described here is ca. 6 Euro!
Check the quality of your RNA by gel to be sure it is not degraded.
- Prepare the annealing mix:
RNA 50 ng/µl 1 µg random hexamers (100 ng/µl) 25 ng/µl 5 µl water add to 20 µl - Anneal the primers in a thermocycler:
1. 70 °C 10 min 2. 25 °C 10 min 3. 4 °C store - Prepare the enzyme mix:
Prepare this mix as a master mix and add then 20 µl to each reaction.5x First Strand Buffer 8 µl DDT 4 µl dNTP (10 mM each) 2 µl SUPERase inhibitor 1 µl SuperScript II 1 µl water 4 µl - Synthesize the cDNA in a thermocycler:
1. 25 °C 10 min 2. 37 °C 45 min 3. 42 °C 45 min 4. 70 °C 15 min 5. 4 °C store - Dilute the reaction mix for use in qPCR
Add 160 µl water to the 40 µl reaction mix.
Use 2 µl per qPCR reaction (that's equivalent to 10 ng RNA).
Random Primer Hexamers:
Dilute 33.3 µl Random Primer Hexamers (3 µg/µl) with 966.7 µl water to obtain a 100 ng/µl solution.
Materials needed:
SuperScript II (# 18064-014) by Invitrogen
Random Primer Hexamers (3 µg / µl (# 48190-011) by Invitrogen
SUPERase Inhibitor (# 2694) by Ambion
Commented Protocol:
1. Prepare the annealing mix:
| RNA | 50 ng/µl | 1 µg |
| random hexamers (100 ng/µl) | 25 ng/µl | 5 µl |
| water | add to 20 µl |
Instead of the random hexamers you can use 4 pmole gene-specific primers or 1 µg Oligo(dT)12-18 (each given as total amount for this reaction mix). I prefer the random hexamers, because one synthesis can be used for whatever you want to detect and is not enriching the 3' ends of the RNA. Furthermore, it just works well.
2. Anneal the primers in a thermocycler:
| 1. | 70 °C | 10 min |
| 2. | 25 °C | 10 min |
| 3. | 4 °C | store |
3. Prepare the enzyme mix:
| 5x First Strand Buffer | 8 µl |
| DDT | 4 µl |
| dNTP (10 mM each) | 2 µl |
| SUPERase inhibitor | 1 µl |
| SuperScript II | 1 µl |
| water | 4 µl |
This works really well. I make a big mix and add it to each tube as soon as the PCR is finished. I just do this at room temperature to avoid the trouble with ice.
4. Synthesize the cDNA in a thermocycler:
| 1. | 25 °C | 10 min |
| 2. | 37 °C | 45 min |
| 3. | 42 °C | 45 min |
| 4. | 70 °C | 15 min |
| 5. | 4 °C | store |
5. Dilute the reaction mix for use in qPCR
Add 160 µl water to the 40 µl
reaction mix.
Use 2 µl per qPCR reaction (that's
equivalent to 10 ng RNA).
For some applications it is necessary to get rid
of the RNA. Use a RNase incubation followed by a clean up using
spin columns.
Known Issues:
- The expensive stuff is the SuperScript enzyme. The rest is supplied in sufficient amounts. If you have less than 1 µg RNA, you can consider to reduce the volume to half and the enzyme to match the amount of RNA. Before you process hundreds of samples try one sample with your optimized protocol versus this standard version.
References and Comments:
This protocol has several roots. It is based on a protocol to create cDNA for microarrays and adapted to be used for lower amounts. It works very reliable and it is my preferred way of cDNA synthesis.
How to cite this page in publications:
This document can be cited like this:
Untergasser A. “cDNA synthesis using superscript II”
Untergasser's Lab. Summer 2008. (include here
the date when you accessed these page).
<http://www.untergasser.de/lab/protocols/cdna_synthesis_superscript_ii_v1_0.htm>.
Please Do Not Reprint This Article:
This article is copyrighted. Please do not reproduce this article in whole or part, in any form, without obtaining my written permission.