What I am doing is pretty straight forward. I am cutting out a fragment with two restriction enzymes. Then enzymes are needed to chew away the two 3' overhangs so I can circulate my plasmid afterwards. I've tried with pfu but with no success in getting colonies after T4 ligation. Could you please help checking my experiment conditions and please indicate any improvements and suggestions? T4 DNA polymerase or Large Klenow fragment?
End-cleaving ingredients: gel purified double digested plasmid 10ul (41.1ng/ul), pfu DNA polymerase from Promega 1ul, 10x Pfu buffer 10ul, H2O 79ul. Incubate at 72℃ for 20min. Purify according to Qiagen pcr purification kit protocol, final step elute in 15ul H2O.
Then I performed T4 ligation: 2x ligation buffer 5ul, 4ul (out of 15ul) eluted fragment, T4 ligase 1ul. Incubate at 4℃ overnight. Transformation and no colonies.
Thanks in advance.
Your reaction mixes looks no problem to me, may I know which method of transformation did you use? And did you prepare competent cells yourself?
If you can locate this paper below, you can see the factors to produce the highest filling-in efficiency by using Pfu.
Biotechnol Appl Biochem. 2005 Dec;42(Pt 3):223-6.
A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase.
Abstract
The present paper reports a highly efficient method of making blunt ends from cohesive ends of double-stranded DNA. Klenow fragment and Pfu DNA polymerases were used to fill in the cohesive ends. Since the transformation efficiency can directly reflect the filling-in efficiency, similar ligation and transformation conditions were used, and the filling-in efficiency was compared with the corresponding transformation efficiency. The results indicate that the filling-in efficiency of Pfu DNA polymerase was 1.96 times that of Klenow fragment and its efficiency was markedly higher than that of Klenow fragment (P
Hi Yuan-Yeu, I looked into it and couldn't find the full-text but I have just sent out a full-text request to the author so hopefully she/he will kindly reply back. However, for my experiment, I need to cleave the overhang rather than filling in so Iam not sure if the optimal conditions are the same in both cases.
Seems to me, it is kind of complicated... the Pfu can go either directions: removing or fill-in (with different conditions?). I have successfully performed blunt-ended ligation, but I filled the sticky ends with Klenow.
@ Theo,
Thanks for the explanation. Let's hope she can get positive results. In my experience, I saw more people using Klenow than pfu for blunting the ends. Did you experience the same thing?
Best
@ Theo and @ Yuan-Yeu, I'm giving a feedback here. sorry for late reply, our uni shut down for the last two days due to storm so I've just got my result. Unfortunately, I still couldn't get any colonies. :( I checked my concentration of eluted DNA prior to T4 ligation and it was about 78ng/ul, which was good. However, I realized I performed T4 ligation at 4℃ overnight, which is not the optimal temperature. So do you guys think I should try to do the T4 ligation one more time at 16℃ overnight? Since I have never succeed in blunt-end ligation before so do you guys think that might be my problem other than end-cleaving not successful? Thanks.
Thanks for your quick reply Theo.
''2X Rapid Ligation Buffer (C671B): 60mM Tris-HCl (pH 7.8), 20mM MgCl2, 20mM DTT, 2mM ATP and 10% PEG. The performance of this buffer depends on the integrity of the ATP.''
At this stage I'll blunt-end digest another available vector and perform T4 blunt-end ligation at 16℃ overnight in parallel with this one as a positive control and see if I can get any success. I'll let you know as soon as i get the result.
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