Since my ligation is not working (no colonies in insert: vector plate) and analyzing my results where we could find that my digestion is just fine and same in case of my transformation (lawn in positive control plate), I think that there's some issue with my ligation expt. One of my friends suggested that I look for white small precipitates at the bottom of ligase buffer at the time of thawing it for use. Is it the case?
You should check this with a control ligation. A reasonable kit would include a ligation-friendly control DNA, like a lambda ladder, that you ligate & run on a gel. If the size increases, then the ligation mix works.
John Schloendorn Hi!
If ATP hydrolysis is the issue with the ligase buffer, would supplementing ATP in the reaction be a possible solution?
Also, would Hind III Digested Lambda DNA work as a ligation control?
Sure, that would help. Although ATP is pretty hard to kill completely. You'd have to have a serious contamination or protocol lapse to make that happen. I think it's much more likely that the sequences you're trying to ligate are just being difficult, like buried in structures, GC rich, or just cursed.
Yes HindIII is fine. You will have to thoroughly heat-inactivate the enzyme so it doesn't chop your ligated fragments up again as soon as they form.
You mean heat-inactivate the HindIII enzyme before setting up ligation reaction?
JFYI.. I am trying to ligate 2 inserts, one is of 0.7KB another 1.5KB into an expression vector. I have tried 4deg overnight ligation and also RT for 1hr as per user manual. I am getting lawn in positive control of transformation but no colonies on insert: vector plate.
Yes. Hmm. Tough luck. I think it's one of those cursed reactions. Makes you stronger in the long run I suppose. Time to learn Gibson Assembly?
Hi John, Yea sure.. Please let me know if I can do this insert ligation using Gibson assembly?
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