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
- - - - -

Can I use Hot Start Taq polimerase for any target?


  • Please log in to reply
6 replies to this topic

#1 lab_microbe

lab_microbe

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 06 January 2011 - 02:07 PM

My supervisor asked me to order polimerase with high fidelity because she will be doing some experiment with molecular genotyping of some bacterial strains, so I was thinking of ordering a Hot Start Taq polimerase, but while reading additional info, I came across a picture where it says that it improves amplification of targets that require hot start. Now, I am confused, can I use it for amplification of any kind of target gene, or only "special"? And what is so special about this genes- they higher GC-content (so it is needed a higher temperature to disrupt the strands), or?

Thank you in advance! :)

#2 ivanbio

ivanbio

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 116 posts
7
Neutral

Posted 06 January 2011 - 02:22 PM

Do not worry about the statement "improves amplification of targets that require hot start". All that means is that because you need a hot start step to activate the Taq enzyme you are likely also helping denature DNA that has strong stability, like high GC-content DNA. In other words, going through a hot start step (3 to 15 minutes at around 94oC to activate your Taq, depending on the brand of Taq you get) creates an environment (high temperature for a few minutes) that helps in the amplification of high-stability DNAs.

Your hot start Taq will work just fine with most templates; it just so happens that a hot start step also leads to DNA denaturation of otherwise hard to amplify DNAs.

Ivan
Carlsbad, CA

#3 lab_microbe

lab_microbe

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 06 January 2011 - 03:26 PM

Do not worry about the statement "improves amplification of targets that require hot start". All that means is that because you need a hot start step to activate the Taq enzyme you are likely also helping denature DNA that has strong stability, like high GC-content DNA. In other words, going through a hot start step (3 to 15 minutes at around 94oC to activate your Taq, depending on the brand of Taq you get) creates an environment (high temperature for a few minutes) that helps in the amplification of high-stability DNAs.

Your hot start Taq will work just fine with most templates; it just so happens that a hot start step also leads to DNA denaturation of otherwise hard to amplify DNAs.


thank you :)




#4 genom38

genom38

    member

  • Active Members
  • Pip
  • 13 posts
2
Neutral

Posted 06 January 2011 - 03:41 PM

Hello,

Have you ever search information about pfu DNA polymerase? As far as I know it polymerizes DNA more accurately than Taq polymerase.

#5 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 06 January 2011 - 03:49 PM

hi, just for sharing:
high fidelity polymerase is not the same with hot start polymerase...
She might mean pfu polymerase which have proof reading capabilities.
high fidelity taq usually is a blend of both pfu and taq together like:

high fidelity taq: Platinum® Taq DNA Polymerase High Fidelity
high fidelity taq with hot start: AccuPrime™ Taq DNA Polymerase High Fidelity

make sure which one suits your application the best.

just my 2 cents.
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

#6 lab_microbe

lab_microbe

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 06 January 2011 - 05:31 PM

Eventually, when I saw all the enhancement (yield, sensitivity and specificity) features that are gained by hot start polymerase, my mind thought it also has high fidelity.

So to make it clear: hot start polymerase is suitable for small yield templates with/out high GC-content and when we have primer-dimer issues, and Pfu is best for high fidelity, because it has the proof-reading activity. :)

Thank you :)

#7 ElHo

ElHo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 90 posts
1
Neutral

Posted 07 January 2011 - 01:02 AM

There also are some polymerases with both hot start and high fidelity!




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

©1999-2013 Protocol Online, All rights reserved.