![]() ![]() Klenow’s 3’ > 5’ exonuclease activity removes the final nucleotide after the fill-in reaction. 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. 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. 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). ![]() You could probably leave out a few of the purification steps- see other posts for further suggestions about this aspect of the procedure.ġ. However, I really only wanted to do it once. Out of 18 colonies I picked (from over 400), all but 1 was correct.Ī lot of this may have been overkill. later suggested that I might have wanted to increase that to 2 hours, but 1 was sufficient. I incubated the ligation reaction at room temp for 1 hour. 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. 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. Finally, I phenol extracted the fragment. 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). 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 then heat inactivated Klenow by heating at 75 C for 20 minutes. I just wanted to be on the super-safe side.įill-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. 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 was lucky that both NdeI and BsrGI can be heat inactivated. I inactivated the restriction enzymes by first upping the salt to 100mM and then heating at 80 C for 20 minutes. I incubated the reaction for 2 hours at 37 C and ran 1 uL to check for completion of the digest. Preparation of vector- I cut my vector DNA with NdeI and BsrGI in a 50 uL reaction containing NEB 4. Also, I didn't care which way my fragment my fragment went in. I would avoid doing a blunt end ligation if you can find compatible enzymes or can add handy cleavage sites by PCR. *Considerations* There simply wasn't another way to achieve the right construct without doing it this way. I read everything I could about the topic on this forum and came up with the following protocol, which, worked really well. I have recently performed a successful ligation using Klenow to make blunt ends. 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! Ones, I had 5 positive colonies in 48 I picked. Ones of them, incubating 15 min, the other one, 4 or 6 h at 25Â☌ both (I inactive the first one at 70Â☌ 20 min to wait for the second one). Two tubes kenow (NEB) treatment using twice that NEB recommends but no more in any NEB restriction buffer and dNTPs at 50 uM. Gel-purification (if I don’t do that, I have some or many colonies in my c(-). Digest my DNA (a good amount), phenol-chlorof extraction (I think it wouldn’t be necessary, but I do as my boss recommends). What I have done with the best results is: I have try many different conditions!!!! Temperature, klenow concentration, incubation time. She says it’s very important a Phenol-Chloroform extraction before and after klenow treatment and she says she incubated at 37Â☌ over night. I have try it many times with very few successful cases.īy boss says she did it many times (I don’ t know). I’m suffering a lot because I have to fill in many different thing (The reason? A long story ). I’m going to talk about my experience…. I don’t think I could help you very much.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |