Hi all, I have XbaI/BamHI digested linearized plasmid. I want to re ligate the same plasmid. Please provide suggestions.
Hi,
What is your objective? You could fill the sticky ends with a polymerase (e.g. Klenow fragment) and use blunt end ligation to close the plasmid. However, I do not believe that this is the answer you are looking for.
Good luck!
I think both enzymes create 5' over hang. So treat the digested product with Mung Bean Nuclease which removes 5' overhang. Then perform blunt end ligation but you will loose both RE sites.
Make a Fill-In reaction with Klenow or PFU, incubate 100 ng of linearized vector, with the buffer, enzyme and dNTPs, at 37 degrees, 15 minutes, then heat inactivate at 75 degrees for 20 minutes. Make a solvent extraction (like chloroform extraction), then an alcoholic precipitation.
In case of 3´ overhang, first incubate without dNTPs and then with dNTPs
Once you resuspend your precipitated DNA, ligate overnight at 4 degrees (add 15% of PEG8000 to the ligation mixture), then transform 4 µL of the ligation, (when you transform, and plate a ligation mixture transformed bacteria, be sure to centrifuge at max speed for 30 seconds to pellet al the bacteria in the recovery LB media, then leave the pellet with 50-100 µL of LB, resuspend and then plate, that is to be sure that you are plating almost all the population of bacteria that could have the correct plasmid ligation)
Good luck
I done Klenow treatment, ligation and transformation. I got my circular self ligated plasmid. After sequencing of my plasmid I got BamHI restriction site. But sequencing result showed loss of two nucleotides of XbaI (TCTAGA) restriction site i.e sequence is-TCTA. My final sequence at the ligation site is (XbaI-BamHI) - TCTAGGATCC.
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