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cDNA synthesis using superscript II

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.

  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
  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
    Prepare this mix as a master mix and add then 20 µl to each reaction.
  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).

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
Prepare this mix as a master mix and add then 20 µl to each reaction.

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:

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.