klenow fill in protocol for making blunt end - (Jun/13/2006 )
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]
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!
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.
I have recently performed a successful ligation using Klenow to make blunt ends. I read everything I could about the topic on this forum and came up with the following protocol, which, worked really well. I hope it helps someone out there!
*Considerations* There simply wasn't another way to achieve the right construct without doing it this way. I would avoid doing a blunt end ligation if you can find compatible enzymes or can add handy cleavage sites by PCR. Also, I didn't care which way my fragment my fragment went in.
Preparation of vector- I cut my vector DNA with NdeI and BsrGI in a 50 uL reaction containing NEB 4. I incubated the reaction for 2 hours at 37 C and ran 1 uL to check for completion of the digest. I inactivated the restriction enzymes by first upping the salt to 100mM and then heating at 80 C for 20 minutes. I was lucky that both NdeI and BsrGI can be heat inactivated. In fact, it really wouldn't have mattered if I didn't heat inactivate them because the blunting reaction wouldn't have recreated their recognition sites. I just wanted to be on the super-safe side.
Fill-in Reaction- I added 2 uL of Klenow (NEB: 3'-5' exo-) and 1 uL of 2.5 mM dNTPs and incubated at 37 C for 30 minutes. I then heat inactivated Klenow by heating at 75 C for 20 minutes.
Isolation and extraction of blunted vector fragment- I gel purified my fragment by freeze-thaw, ethanol precipitated the DNA, and resuspended in 20 uL CIAP buffer (NEB 3). I added 1 uL CIAP and performed a double heat (15 min @ 37 C, 15 min @ 56 C, add an additional 1 uL CIAP, repeat). Finally, I phenol extracted the fragment.
Preparation of fragment to be inserted- I digested the DNA with PvuII (creates blunt ends), did a squeeze-freeze to isolate the correct fragment, and phenol extracted the DNA.
Ligation- Pretty standard, I included PEG in my ligation mixture, had my fragment in 3-fold excess to my cut vector, and did a self-ligation as a control. I incubated the ligation reaction at room temp for 1 hour. My P.I. later suggested that I might have wanted to increase that to 2 hours, but 1 was sufficient. Out of 18 colonies I picked (from over 400), all but 1 was correct.
A lot of this may have been overkill. However, I really only wanted to do it once. You could probably leave out a few of the purification steps- see other posts for further suggestions about this aspect of the procedure.
1. Best buffer: 10x Klenow buffer: 500 mM Tris-HCl, pH 7.4, 100 mM MgCl2, 1 mM dithiothreitol (DTT) and 500 µg/mL Bovine Serum Albumin (BSA). Mix 5 mL of 1 M Tris-HCl, pH 7.4, 1 mL of 1 M MgCl2, 10 µL of 1 M DTT and 500 µL of 10 mg/mL BSA with 3.5 mL of Type I water. Aliquot and store at -20°C. Discard if precipitated.
2. C. Joyce cautions that the use of more enzyme, longer reaction times than 10-15 minutes, or temperatures higher than room temperature which promote breathing may decrease the yield of filled-in product. Klenow’s 3’ > 5’ exonuclease activity removes the final nucleotide after the fill-in reaction. Alternating polymerase and 3’ > 5’ exonuclease activity eventually convert all of the terminal dNTP to dNMP which results in the loss of the terminal nucleotide. The exo- version of the enzyme is not recommended for fill-in reactions prior to DNA ligation, as extra nucleotides added to the 3’ end of a blunt-end DNA will not be deleted by the exonuclease activity of the intact enzyme. (C. Joyce, 1984 BRL Focus 6 (1) 6.)