I am getting repeated failure in cloning of large insert into small vector. I am ligating a cut insert of 7741 bp to a cut vector of 4312 bp. The enzymes used are SPEI and HINDIII in insert and SPEI and NOTI in vector. Hindiii site in insert and NotI site in vector are blunted. So it’s one end cohesive and other end blunt ligation. I have tried all ligation ratios, was able to get colonies after ligated product transformation but all proved to be false construct. The size of the false construct plasmid (linearized) is around 3kb.
Kindly guide me what to do. ??
Thanks Dr. Hideyo Yasuda. I am doing exactly what you have mentioned. The experimental steps are attached here.
Hi,
I was facing a very similar problem while trying to ligate a 12.5kb insert with a 3.8kb vector. Both of the vector and the plasmid have a common cohesive end and a blunt end. I learnt that DNA fragments tend to circularize under relatively high temperature. so how about try a 4℃ ligation for 1-2 days to replace the traditoinal 16 ℃ or 25℃. also, make sure the inserts molar ratio are high enough!!
best wishes
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