BamHI/XbaI cloning, bad combination? - (Nov/30/2005 )

Hi there,
Im having problems cutting a plasmid with BamHI/XbaI. I get a lot of colonies in my ligation control meaning I have incomplete cleavege. Repeated already three times (different conditions) and I always get lots of uncut plasmid. I checked the nts following the XbaI site and they dont build the dam site. Some time ago a heard from someone here having the same problem with XbaI/BamHI. If methylation is not the problem here, then what could it be?? Thanks a lot for ur reply!!

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hi
what is the buffer you use? is it a double digest or a 2step digest?
do you dephosphorylate your vector?
Ho do you purify it?

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At first I used BamHI for both, then I realised I should do a 2 step digestion. Did the first (with BamHI, 3h, 37°C), purified with qiagen kit, then did the second with XbaI-NEB2, ON at 37°C. Gel purified, extracted and ligated (1h, RT). I skipped over the dephosphorylation this time...A got fewer colonies in the control, but even less in my ligation. What would u suggest?

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hi
i would suggest an overnight digestion in NEB buffer 2 + BSA (i've done it with no star activity over 2 days, so ovenight 37° is ok.
then a gel purification

if that doesn't give more results, you would dephosphorylate. (you can do 2 assays in same time but for efficient dephosphorylation dna is quite very diluted so that increase sample for gel purification...)

in the gel extraction procedures, you may have in the protocol procedure for sensitive fragments that are used in ligations (second column wash or let sit the washing buffer 5' prior to spin... etc)
i got better yields when do them.

finally ligation ime can be increased, and i've succesfully done overnight at RT or 14 to16°... For secure, i let at least 2h.

keep informing.

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