Ligation issues
Lalitha Srinivasan
Friday, 27 February 2009 15:06 UTC
I am trying to ligate the 1.2kb Kan cassette from pUC4k into a pBluescript vector containing my sequence of interest. I digest both plasmids with EcoRI, run the digested products on the gel, gel purify my DNA & set up the ligation. I use T4 ligase from Invitrogen and follow the benchtop ligation protocol. Vector:insert = 1:3. I do not get any colonies on Lb+kan plates, though I get few colonies on LB+carb plates (pBlue carries the Amp resistance gene).I have tried treating the vector with CIP/antartic phosphatase, checked my LB+Kan plates by plating out the pUC4k E.coli. Tried out self ligation reactions which work well. Nothing seems to work at all.
Any suggestions??
Lalitha
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Replies
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There are several things that may be going wrong here. You will need to have controls for every-single-step.
When cutting with a single enzyme, don’t “try” to CIP – ALWAYS CIP.
Check your Antibiotic concentration, is it too high?
Check your DNA concentration, is it too low? V:I ratio 1:3 is good, but it is no good if the amount of vector DNA is 5 ng.
If pUC4k Kan cassette has Kan under prokaryotic promoter, always use Kan-LB plate. That itself will give you correctly cloned colony, because insert is not supposed to have ori sequence.
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Doing the ligation over night at 4ºC might also help.
Good luck
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