Problems facing in cloning with pBI121 vector - (Oct/31/2009 )
I tried to clone four genes in pBI121. out of which two got transformed and im facing the prob with other two... i used BamH1 and Sma1 for transformation. the size of the genes tht got cloned is 250bp and the size of rest two is 615 and 850bp respectively... The size of vector is 13kb. My doubt is ... Do large size inserts need more restriction time than small size inserts. i even did tried with 16hr incubation with each enzyme sequentially... but it didnt work... kindly help..
Why do you think the problem is the restriction digest? We need MUCH more information to give any sensible answer.
Why aren't you using BamHI and XbaI as enzymes? A mixed blunt/cohesive end ligation would not be where I would start.