Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

cDNA convertion to DNA


  • Please log in to reply
4 replies to this topic

#1 aidaelshaer

aidaelshaer

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 01 October 2010 - 03:37 PM

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.


#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 02 October 2010 - 04:52 AM

- 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

#3 aidaelshaer

aidaelshaer

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 02 October 2010 - 06:31 AM

- 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


#4 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
110
Excellent

Posted 04 October 2010 - 04:49 AM

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.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 aidaelshaer

aidaelshaer

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 04 October 2010 - 03:51 PM

Thanks alot Dr Veteran for your answer.

Aida

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





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.