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
- - - - -

PCR with pfu and degenerate primers (?)


  • Please log in to reply
7 replies to this topic

#1 Sciurus

Sciurus

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 26 March 2009 - 07:00 AM

Hi all,

Hopefully, someone can help me!
I want to blunt end clone some pcr fragments but I'm stuck at performing the pcr with pfu polymerase. Because the sequences of the genes I'm looking for are unknown, I am using degenerate primers (but they don't contain inosine). Pcr with taq worked fine - nice bands and everything.
Unfortunately, my primers won't be efficient for TA cloning. Thus, I ordered the Stratagene blunt end cloning kit which already contains pfu polymerase.
But every attempt at getting my pcr fragments with pfu failed so far. I tried the recommended protocol from Stratagene, as well as my optimized protocol from pcr with taq - none produced any bands (not even smear).

Maybe someone knows a trick or something that could get me my bands!
I'll be eternally grateful ;-)

#2 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 26 March 2009 - 08:10 AM

Hey,

How about taking the Taq PCR product and using it as the template for pfu reaction? This PCR should be easy.

Best,
TC

Hi all,

Hopefully, someone can help me!
I want to blunt end clone some pcr fragments but I'm stuck at performing the pcr with pfu polymerase. Because the sequences of the genes I'm looking for are unknown, I am using degenerate primers (but they don't contain inosine). Pcr with taq worked fine - nice bands and everything.
Unfortunately, my primers won't be efficient for TA cloning. Thus, I ordered the Stratagene blunt end cloning kit which already contains pfu polymerase.
But every attempt at getting my pcr fragments with pfu failed so far. I tried the recommended protocol from Stratagene, as well as my optimized protocol from pcr with taq - none produced any bands (not even smear).

Maybe someone knows a trick or something that could get me my bands!
I'll be eternally grateful ;-)



#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,501 posts
252
Excellent

Posted 26 March 2009 - 08:19 AM

Why can't you extend the 5' end of your degenerate primers to that they are efficient for TA cloning? You could blunt the ends of your Taq amplified sequence and clone it with a blunt ligation, but this is probably more difficult.

#4 Sciurus

Sciurus

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 26 March 2009 - 08:36 AM

First of all thanks for your replies! :rolleyes:

@T C

What would the difference be if I used the taq fragment instead of cDNA for pcr success?


@Phage:
Most of my primers end with C, so there probably won't be many A-overhangs.

Do you mean adding extra A residues to the 5' ends of my primer? Post pcr? Could you explain how that works?

How would I blunt the ends of my taq products?

#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,501 posts
252
Excellent

Posted 26 March 2009 - 06:04 PM

The extra A is added to the 3' end of the PCR fragment, and will be far away from the final 3' base of your primer. (either one). I thought you were referring to the (small) effect of the 5' primer base on the addition of the 3' A on the opposite strand.

You can blunt a PCR fragment with, e.g. the Epicentre End-It DNA repair kit, for example.

#6 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 26 March 2009 - 07:28 PM

Hey,

I was under an impression that you were using genomic DNA as template. It is my personal experience that PCR using a clone or a PCR fragment is much easier than doing a PCR using genomic DNA as template and I use pfu.

I am not sure if there will be any difference b/n cDNA and taq fragment but its worth a shot. Do tell me if you do it and if it works. :wacko:

Best,
TC

#7 Sciurus

Sciurus

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 26 March 2009 - 10:48 PM

The extra A is added to the 3' end of the PCR fragment, and will be far away from the final 3' base of your primer. (either one). I thought you were referring to the (small) effect of the 5' primer base on the addition of the 3' A on the opposite strand.


Sorry, I guess I didn't express it right in my earlier post. I meant that my primers start with a G, so that the pcr fragment will end on C. From what I read I got the impression that TA cloning would be inefficient if having such fragments because most of them would have a G overhang instead of A...

Hey,

I was under an impression that you were using genomic DNA as template. It is my personal experience that PCR using a clone or a PCR fragment is much easier than doing a PCR using genomic DNA as template and I use pfu.


I used both genomic and cDNA but none produced any fragments. But I will try another pcr with the taq template.
Have you ever used pfu in combination with degenerate primers? Do I maybe have to make some special adjustments because of that? I couldn't find anything about it except that the primers mustn't contain inosine - which they don't...

Edited by Sciurus, 26 March 2009 - 10:49 PM.


#8 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 27 March 2009 - 02:31 AM

Hey,

Nope......I have never used pfu with degenrate primers. :)

Best,
TC




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

©1999-2013 Protocol Online, All rights reserved.