I want to clone a 7 kbp pDNA with a 1.2 kbp fragment and I have already tested the 1: 3, 1: 1 and 3: 1 ratios but I always get self-ligation of the vector. How can I resolve this or improve the cloning? Thanks
Hi Joel,
Could you give us some more detail on your procedure?
I suppose you are cloning using restriction sites. Is your fragment derivingfrom a enzymatic digestion? Did you dephosphorylate your vector?
Piero
Hi Piero,
Thanks, yes i can. I'm cloning using cohesive restriction sites with XbaI and BamHI restriction enzymes. No, i don't dephosphorylate your vector yet.
You need more details?
Joel
" I always get self-ligation of the vector" ... adding the dephosphorylation step could help to lower down the background.
One more question, your insert come from a PCR reaction?
Yes i know, i didn't have this resource, but i will try soon as possible. Yes my insert come from a PCR reaction but i do a purification of PCR
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