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
- - - - -

RT-PCR - dilution cDNA prior to PCR?


  • Please log in to reply
4 replies to this topic

#1 drdtkk

drdtkk

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 02 September 2004 - 04:44 AM

Hi
I wonder could you answer a basic question for me: Having performed 1st strand synthesis do I have to dilute the cDNA prior to PCR analysis. I designed primers and am using PCR to amplify the product before electrophoresis but I am confused as to whether I need to dilute the cDNA prior to performing the PCR reation. In the promega protocol it says to dilute to 100ul but in others it doesn't mention it.

thanks
Dtkk

#2 phegene

phegene

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 02 September 2004 - 08:33 AM

If your target is a rare message, I would not dilute the cDNA before PCR, however if it is really abundant...diluting would be better for the PCR reaction. If you have enough sample I would try both ways and see which one gives you the best yield.

#3 mario2004

mario2004

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 03 September 2004 - 05:41 PM

I agree.

I didn't do dilution until I found the protocol coming with the promega kit, then I think diluting RT products is good in two ways: giving you more templates and making detecting subtle expression changes easier.

#4 beth

beth

    member

  • Active Members
  • Pip
  • 14 posts
1
Neutral

Posted 09 April 2009 - 07:16 AM

You MUST always dilute your cDNA before performing PCR, because the reagents in the RT reaction can interfere with the reaction. It will tell you how much you need to dilute out your cDNA in the protocol of your RT kit.

#5 Ahrenhase

Ahrenhase

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 146 posts
10
Good

Posted 09 April 2009 - 08:17 AM

Here's an example of what I do and it seems to work out rather well.

I use ~.5 ug RNA for my cDNA synthesis (can use b/w 50ng-5ug i think)
I use a First Strand Kit (superarray biosciences) and after the reaction I dilute the 20ul reaction into 91ul H20 (as it tells you to do in the protocol)
If the reaction is 100% efficient, which it's most likely not, this will give me a concentration of 45ng/ul
For my reaction I use 15ul template cDNA (~675ng if reaction is 100% efficient)

I use 18s rRNA as my reference gene and it is relatively abundant (~8 Ct value @ 675 ng), but my lower expressed genes show up too (~30 Ct)




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

©1999-2013 Protocol Online, All rights reserved.