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hi,
could somebody give me protocol for making blunt ends with klenow.
I tried several times by incubating at 25 degree for 15 min , then in second attempt at 37 degree for 15 min and later in another attempt, 37 degree for 20 min then heat inactivated at 70 degree for 20 min.I used T4 DNA ligase buffer for klenow and added 33uM each of dNTP at final conc. additionally.
Later I added 3U of T4 DNA ligase and kept for ligation at 25 degree for 4hrs. A tube which I kept as control got ligated but klenow filled did not. The starting DNA for fill in was pEGFP-N1 digested with Nhe I and Eco RI.
could some tell me how make it work
thanks in advance[color=#003300]

Hi guy,
I don’t think I could help you very much. Any way
I’m going to talk about my experience….. I’m suffering a lot because I have to fill in many different thing (The reason? A long story ). I have try it many times with very few successful cases.
By boss says she did it many times (I don’ t know). She says it’s very important a Phenol-Chloroform extraction before and after klenow treatment and she says she incubated at 37°C over night.
I have try many different conditions!!!! Temperature, klenow concentration, incubation time.
What I have done with the best results is:
- Digest my DNA (a good amount), phenol-chlorof extraction (I think it wouldn’t be necessary, but I do as my boss recommends). Gel-purification (if I don’t do that, I have some or many colonies in my c(-).
-Two tubes kenow (NEB) treatment using twice that NEB recommends but no more in any NEB restriction buffer and dNTPs at 50 uM . Ones of them, incubating 15 min, the other one, 4 or 6 h at 25°C both (I inactive the first one at 70°C 20 min to wait for the second one). Then, phenol-chlorf extraction again.
- Ligation at 14°C overnight.
- Then I pick up 50 colonies. If I don’t have a good one. I pick 50 more.

Ones, I had 5 positive colonies in 48 I picked. Ones, I had 1 positive colony in 50. Ones 1 in 72 colonies and many times I just have nothing !! I wish someone could give me some good advices or show me how to do it with better results!

Good luck!!

I have used "DNA Polymerase I, Large (Klenow) Fragment" from NEB. They have a protocol. Worked fine.

It worked for me using 0.25mM dNTPs, 15 min at RT.