I am trying to clone a 1.3 kb fragment using the pJet system. It is a sticky end cloning protocol and I have tried different ratios (from 3:1 to 8:1). I have used DH5a and Top10 cells with transformation controls and transformation does not seem to be the problem. Also, I tried longer ligation time (30 min) and I get no colonies. Please help!
Hi Alejandra Puentes
I think it is better that you check the quality of both the vector and the insert fragments on an agarose gel, before doing the ligation. It is better to clean both RE digested products using a DNA Cleanup or PCR Cleanup kit. Sometime, you may have a good number from Nanodrop with good ratios, but you might not have the fragment that you are interested in.
Using NEB T4 ligase, I incubate the ligation mixture (1vector: 5 insert) for 30min-1h at room temperature and do the transformation. It always works with me.
Cheers
Thank you all for your recommendations. I already got my clones! The problem was the PCR reaction. We used different primers and a high fidelity polymerase :)
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