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
- - - - -

Hotstart PCR and unspecific Amplification


  • Please log in to reply
3 replies to this topic

#1 Nebulus

Nebulus

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 20 October 2010 - 01:41 PM

Hi Everyone,

I am using 5 species specific primers allocated to 2 different multiplex reactions to screen the guts of predators for prey remains.

I optimized my PCR about a year ago using Invitrogen's PCR optimizer Kit. I found the optimal conditions did not cause unspecific amplification for any of the 5 species.

I decided to try 5-Prime's Hotmaster PCR Taq polymerase with the associated 5X Buffer to see if I could increase the sensitivity and yield of my reactions. I got a major increase in desired band intensity and sensitivity, however using this buffer and special Taq polymerase I am getting major unspecific amplication from most of the primer sets.

I thought Hotstart PCR mixes were supposed to increase specificity due to the Hot start and Cold Stop enzyme activity.

The only thing I could think of is that the buffer has a "release when needed MgCl" function. I know increased MgCl decreases specificity...


Any thoughts?

#2 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 20 October 2010 - 03:33 PM

I not sure about the "release when needed mgcl" function, but are you sure it is 5x?
Based on http://www.5prime.co...polymerase.aspx
it says it is 10x buffer...

but then, we need to know regarding the unspecific is the longer product or shorter product.

My experienced when switch to other hotstart taq especially those fast amplification rate polymerase like Kappa Biosystem, KOD hotstart and phusion flash, I have to re-optimize again and is not just as easy as "just replace the polymerase and maintain the other formulations".
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 Nebulus

Nebulus

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 20 October 2010 - 04:00 PM

Sorry Yes it is 10x and I have been using it as 10x. My old PCR buffer was 5x, so all I changed in the protocol was replace standard Taq with hotmaster Taq and use 5ul of the hotmaster buffer instead of 10ul of my old 5x buffer and increase the water by 5ul to compensate for the volume change.

Not sure what you mean by the longer or shorter product. For example multiplex reaction #1 has 3 primer pairs that screen for 3 species, while multiplex reaction #2 has 2 primer pairs that screen for the other 2 species. Before using hotstart, multiplex #1 only amplified the species it was supposed to. Now some of the primer pairs in that multiplex are amplifying the DNA of the other 2 species (gives bands of expected size of species "A" it is supposed to test for when DNA of species "B" is present when there should be no bands).

#4 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 20 October 2010 - 08:51 PM

if that is the case, you just have to re-optimize your PCR again, with new primers set. As I mentioned earlier, based on my experience when I change taq, all the condition changes as well. The non-specificity might due to unspecific binding, or primers contamination.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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

©1999-2013 Protocol Online, All rights reserved.