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second strand cDNA - (Aug/19/2004 )

Do you have a second strand cDNA protocol?
Please, share with me.
(There are a loads of first strand kit at manufacturers, but I never find second strand.)





I guess no protocol is needed as you obtain first cDNA strand, you can use that first cDNA as template in PCR reaction. Do it as a normal PCR. It works.


I need second strand DNA, because after sythesis I would like to digest by RE.

Thanks stratagene web site.


also you use this protocol,

- First strand cDNA synthesis
Add together
20 µl poly(A)+ RNA (~1.0 µg)
1 µl oligo-dT25-bio (700 ng/µl)
4 µl H2O
Then add
8 µl 5x First Strand buffer
4 µl 0.1 M DTT
2 µl 10 mM dNTPs
1 µl Superscript II (200 U/µl)

Incubate 2 hr at 42°C
- Second strand synthesis
Add together
40 µl first strand reaction mixture
32 µl 5x Second Strand buffer
3 µl 10 mM dNTPs
6 µl 0.1 M DTT
15 units E. coli ligase
50 units E. coli polymerase I
1.6 units RNAse-H
H2O to 160 µl
Incubate 1 hr at 12°C and 1 hr at 22°C
Extract the reaction mixture once with phenol/chloroform and purify the cDNA using a Qiaquick spin column (qiaquick PCR purification kit, qiagen). Check the quality and yield of cDNA by agarose gel electrophoresis.
5x First Strand buffer:
250 mM Tris.HCl pH 8.3; 15 mM µgCl2; 375 mM KCl

5x Second Strand buffer:
94 mM Tris.HCl pH 7.0; 23 mM µgCl2; 453 mM KCl

750 mM NAD+; 50 mM (NH4)2SO4

First Strand buffer, Superscript II, E. coli ligase and DTT: Gibco-BRL.
E. coli polymerase I, RNAse-H and the dNTPs: Pharmacia.

500 ng of the double stranded cDNA preparation is used for AFLP.

good luck
Hassan soltanloo