We are using pET-32 Xa/LIC kit (Novagen 70072-3) for cloning. T4 DNA polymerase given with the kit to generate compatible overhangs on the insert. After T4 DNA polymerase treatment we we have annealed the insert with the Xa/LIC vector. Then we have transformed it into DH5α competent cells (Hi media) but no colony was found.
Is it possible that overhang is not properly produced on insert because we have not stirred T4 DNA polymerase in the vial before adding it into the reaction mixture?
So many things can happen that will prevent colonial growth. The polymerases provided in kits are usually very concentrated, so even though it should have been stirred before pipetting for reproducibility and performance, I doubt that this would be the reason why NO colony was found.
Did you try to migrate your vector and inserts on agarose gel to check if they have the proper lengths before/after annealing? That would tell you if the problem lies in your vector creation or in the transformation/growth of the cells.
You do not have to mix enzymes. Just short spin if there are droplets near the cap. If there are insoluble precipitates, do not use the vial.
As for your experiment, are your competent cells fine ? Did you check their efficiency with a positive control plasmid ?
Praveesh Valissery Thank you sir for your reply.
We are getting colonies of test plasmid it suggest that our competent cells are fine. But there are no colonies of positive control insert (given with the kit) as well as our desired target insert.
Then is there any problem during annealing?
Myriam Labbé Thank you ma'am for your reply.
This is a ligation independent cloning. Yes ma'am, we performed agarose gel after annealing reaction and we found three separate bands of annealed sample.
One is near 3000 bp, which is the size of the positive control insert, second one is near 6000 bp which is the size of the vector and the last one is found just above 8000 bp.
What does this result suggest ma'am?
Then I don't think the T4 is the problem and your vector+insert seems fine to me.
Something else is preventing the growth of the cells that have received vectors with inserts. I do not know this specific kit, but you might want to look more into its expression mechanism.
Yeah, if you are seeing the annealed 8kb band at better intensity than the vector and insert bands, then there isn't problem with the enzyme. Although you can enrich the annealed clone by varying the vector : insert ratios (1:3, 1:5, 1:7).
And if the competent cells are viable, then the problem could be with the clone that your are getting. Is this an expression vector ? If so, then leaky expression of your insert could be toxic to the cells.
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