I have two different construct:
1. Insert 1 (~3kb) at XhoI and ApaI site in NCVB vector (9.7 kb).
2. Insert 2 (~1.7kb) at XhoI and ApaI site in in pENTR/D vector (2.6kb)
I want to replace Insert 1 with Insert 2. I have already tried restriction digestion followed by Quick ligase (NEB) or T4 ligase (Thermo). I even tried using CIP as I got a large number of self-colonies without it. However, upon CIP usage, no colonies were seen in either plates (self and test). Kindly suggest ways to go about this cloning. The final product that I want is
“Insert2 in NCVB vector at XhoI and ApaI sites”.
PS: I am using ultra-competent DH5a (CSHL protocol) for all my cloning.
If you gel purify your vector and the desired insert then you should mostly get the clone you want. Did you confirm that you are getting complete digestion of the vector construct? If you even have a tiny fraction of uncut or singly cut plasmid then that will swamp out the thing you want upon transformation.
You may try to use different vector to insert ratios.
Please see also:
https://www.researchgate.net/post/Which_ratio_of_vector_to_insert_do_I_need_to_use_in_the_ligation_reaction
https://www.researchgate.net/post/What-is-the-minimum-concentration-of-vector-and-insert-DNA-to-start-a-ligation-reaction
Michael J. Benedik I do not see any uncut or single cut bands in the agarose gel. I will run the gel for longer this time such that possibly I don't get any uncut plasmid contamination.
Mohammad Sadegh Taghizadeh That is a possibility which is unlikely but possible. Next time when I do it, I will use enzyme from different tube. Let us see if that solves the issue. Thanks!
There was an internal XhoI site in the vector which was creating issue. I got the clone by using different sites and another shuttle vector. Thanks everyone for their valuable suggestions.
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