Hello everyone,
thesedays i have serious problem in cloning.
I usually do T-vector cloning with insert before I ligase into expression vector.
T-vector cloning is comfirmed by sequencing but the problem is that when I ligase the insert into the vector, only one nucleotide in enzyme site is always cut by sometihing.
I tried to do again and again, and also use another enzyme, but the problem occured with same.
I used EcoR1, HindIII and BamHI and problem site(Bold) is following,
GAATTC, AAGCTT, GGATCC
I thought 2 possibilties, one is star activity and the other is mutation by UV during gel slicing or....something.
But to reduce star activity, I cut them in short time (2-3hrs) and used enzyme exclusive buffer. and I use NEB enzyme.
Dose anyone have like this problem? or If u have good idea, please give me ur suggestion about my ploblem, thanks alot.
ligation 부터 다시 하는게 어때? ㅋㅋ
내가 최근에 cloning할때 비슷한 상황이 생겼었어.
그래서vector랑 insert두 다 새로 얻어서 다시 ligation했어.
그랬더니 됐어.
처음에 vector랑 insert 얻을때 enzyme cutting에서 잘못됐던지 아니면 gel slice얻을때 잘못됐던지 암튼 처음에 얻었던 vector랑 insert가 문제 있었던거 같애.
제 경험담은 여기까지입니다.
ㅋㅋㅋ
Hi there,
Contamination with an exonuclease activity may explain the results.
Are you 100% sure of the sequencing data? Sometime doublets are not properly resolved...
Could you increase the distance between your EcoRI / HindIII / BamHI restriction sites and the end of the primers used for amplification? This would then give space at the ends that would firstly mean that any exonuclease activity might still leave the restriction sites intact, and secondly, would maximise the efficiency of digestion. It could also mean that you could attempt direct digestion and sub cloning of the sequence into your final vector in one step.
Good luck.
Hi Garam Wi
I agree with Dominique Liger answers ..good luck.
You can switch to the Gibson Assembly from NEB. It's efficient and you no longer need to deal with the restriction enzymes issues.
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