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
- - - - -

amplification of 14Kb


  • Please log in to reply
1 reply to this topic

#1 harry348

harry348

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 71 posts
3
Neutral

Posted 16 July 2012 - 03:56 AM

Hello All,

I need help from you to amplify a 14kb insert.
I have been trying to amplify this using TAKARA LA Taq(Long amplification taq) with different optimization steps of annealing temperature,cycles,Mg+ and so on.
but i could get only a smear Posted Image I also tried with different polymerases but in vain Posted Image

Is there any other possible way to amplify.
like breaking this whole 14kb into 2 or 3 parts ,amplify separately and later join them and then use them finally for transformation?

Any suggestions will be happily received.

Thanx in advance.

harry

#2 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 16 July 2012 - 07:23 AM

Is your target gene GC rich? IF yes, you might consider add DMSO, BSA or Betaine as addictives.
Also, can you describe your PCR conditions and mastermix in detail? It will be very hard for us to troubleshoot for you without sufficient information.
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.