Klenow or T4 PNK to fill in? - (Mar/11/2005 )

First time posting here.

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?

what's your purpose of cloning this 6kb insert into the PUC19 vector? just for regular cloning or try to express it? i am not sure whether PUC19 is expression vector or not. i think if your purpose is to clone your fragment, you can generate one of the restriction enzyme site in the 3' end of your fragment. but you must make sure that there's no such an restriction enzyme site amony your fragment. another point i could figure out is that your fragment may be too long. sorry i never did blunt-end ligation before and don't know much about the difference between knelnow and T4 DNA POLYMERASE. GOOD LUCK.

The ultimate purpose is to get the construct for conditional K/O of a gene. pUC is used as an interim vector. Once the frgmt is in, I can use the cloning sites on pUC to cut it out and insert it to the target vector.
Thank you for your reply anyway. Hope somebody can help me.

hi
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
Fred

Lost my reply somewhere...
Thanks fred 33 for your help. I will have to start from scratch if it doesn't work after a few attempts.

T4 PNK does not fill-in. T4 Polynucleotide kinase transfers the g-phosphate from ATP and other nucleoside triphosphates to the 5'-OH ends of DNA, RNA and 3'-phosphomononucleotides. This would allow previously dephosphorylated vector to ligate to itself.

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.

Thanks tfitzwater! I will try with T4 DNAP.

I was reading your post and I really hope you could answer my question.

I am trying to put an 6 kb insert into pBluescript by blunt end ligation, pB cut by EcoRV.
BUT I don't get ANY product, so I really want to know how you did it. Could you tell me exactly what you use with your ligation?

It shouldn't make a big difference to use a pBluescript in stead of PUC19 right?

I hope you can answer me,
Thanks in advance

Evelyn