4 replies to this topic

[aidaelshaer]

Hello every one ...
i extracted RNA from human cell line.and converted it in to cDNA (using two steps TR Kit.
now iam goning to do conventional PCR reaction for detection specific gene expression.
so iam asking about:
First Quistion:
do i need to convert cDNA in to double strand before pcr ?
or immediatly run the pcr reaction with single stranded cDNA?

Second Quistion:
what is the time needed for the pre-denaturation step for cDNA in conventional PCR?
and is it differ according to the source of RNA?

and if it is a single strand cDNA , i think that the pre-denaturation step in negligble or what??

thanks alot

Edited by aidaelshaer, 01 October 2010 - 03:44 PM.

[Curtis]

- what you have is a first-strand cDNA. you can run PCR with it directly, no need to synthesize second strand
- normally 95C for 3 min is enough, but it depends on the polymerase that you want to use too. check the data sheet of your polymerase.
- your source of RNA is human sell line, it does not differ. follow the datasheet protocol
- you still need to dentarure your cDNA

[aidaelshaer]

Curtis, on 02 October 2010 - 04:52 AM, said:

- what you have is a first-strand cDNA. you can run PCR with it directly, no need to synthesize second strand
- normally 95C for 3 min is enough, but it depends on the polymerase that you want to use too. check the data sheet of your polymerase.
- your source of RNA is human sell line, it does not differ. follow the datasheet protocol
- you still need to dentarure your cDNA

thanks alot Metaller scientist for ur answer,,

iam using DreamTaq Green Master mix from Fermentas.
but how it depend on the polymeras?
and why i still need to denature cDNA , while it is a single strand?
i will be very thankful if u answer me with some details.

Thanks alot.
Aida

[Trof]

There are some polymerases that require longer denaturation step, because they're originaly inactivated by antibody or some modification and longer denaturation degrades the inhibiting agent thus activating the polymerase. But this is not the case with your polymerase, it has recomended denaturation 1-3 min.

When cDNA is created it's a duplex of original RNA with newly syntetized strand. Some RT kits leave the product like that, so you actually have RNA-cDNA heteroduplex. Some RT kits contain RNase H that degrades the RNA after the duplex is syntetised, so leave only single strand cDNA.
Even in that case the single strand creates secondary structures like hairpins and connects to other partialy complementary parts, so that denaturation is needed.

[aidaelshaer]

Thanks alot Dr Veteran for your answer.

Aida

Edited by aidaelshaer, 04 October 2010 - 04:50 PM.