Klenow or T4 PNK to fill in?
Posted 11 March 2005 - 11:51 AM
I read the pinned thread re: blunt-cloning. Very useful.
Now my question is: Which enzyme to choose to fill in?
I picked up somebody else's cloning project to continue. This is a 6kb insert, 5'- end of it is ligated onto the BamHI site of pUC19, the 3'- end of the insert is not compatible with the vector cloning site. So I have to fill in the two ends, and do a blunt-ligation to complete the circle.
The insert has a 3' overhang of 4bp, and the vector, 5'- overhang of 4bp. I tried Klenow and T4 PNK. Hardly any clones for the Klenow one (my enzyme is a bit old too), and lots for T4. But when I checked a few clones from the T4 by digestion, all are pUC only.
Anyone can give me some suggestions?
Posted 11 March 2005 - 12:51 PM
Posted 11 March 2005 - 06:36 PM
Thank you for your reply anyway. Hope somebody can help me.
Posted 13 March 2005 - 06:28 AM
i was wondering : is oit possible to make an adaptator for your vector in which you,may insert the right restriction site for your 3' fragment ? it could be a Bam HI-site of interest-Bam HI adaptator (2 phosphorylated oligos should be ok then annealing).
May i suggest you an other one : by a PCR is it possible to choose ends coompatible with our vector?
Hope i help you
Posted 15 March 2005 - 08:35 AM
Thanks fred 33 for your help. I will have to start from scratch if it doesn't work after a few attempts.
Posted 15 March 2005 - 01:19 PM
T4 DNA polymerase or Klenow Fragment can be used to fill-in 5' overhangs. They both have 5' > 3' polymerase activity. T4 DNAP has strong 3' >5' exonclease activity in the absence of dNTPs. If you briefly treat the DNA with T4 DNAP without dNTPs, both 3' ends will be chewed back. Then add dNTPs and they will be filled-in and create the blunt ends you need.
Edited by tfitzwater, 16 March 2005 - 07:19 AM.