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
- - - - -

should I do blunt end my pcr product before ligation

ligation pcr

  • Please log in to reply
2 replies to this topic

#1 gulsatar

gulsatar

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 June 2012 - 05:53 AM

Hi friends

I have pBluescript vector and e coli which I made by myself. I used Onetaq (NEB) for my pcr reaction. I used degenerate primers and I dont now full sequens of my sample, neither enzym cut site.
My questions

1. should I do any treatment to my pcr product before ligation
2. I dont know enzym cut site of my sample. If I use EcoRV for blunting end. Does it work.
3. If I directly ligate my pcr product to vector. what happens. Does it work


Thanks

#2 Papaver

Papaver

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
19
Good

Posted 07 June 2012 - 11:14 PM

Hi,
do you know if the PCR-product is already blunt end? The common Taq polymerase produces an A-overhang, other Polymerases not (like Phusion). I once used this for blunt end cloning into a blunt cutted vector.

#3 Kamran

Kamran

    Kamran

  • Active Members
  • PipPipPipPipPip
  • 41 posts
2
Neutral

Posted 08 June 2012 - 01:30 AM

Q8: What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?

A8: A sufficient proportion of PCR products generated using OneTaq® DNA Polymerase contain dA overhangs at the 3´end to enable ligation to dT/dU-overhang vectors.
http://www.neb.com/n...tM0480.asp#2167



The best option is to TA clone your PCR product however, you may also do blunt end cloning after phosphorylating your inserts termini (if you are not using phosphorylated primers)




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

©1999-2013 Protocol Online, All rights reserved.