Ligation problem - (Oct/08/2005 )
I digested my vector with EcoR1 and Xmal1 overnight and gel purified the vector. The insert was amplified by PCR and after gel purification I digest the fragment with EcoR1 and Xmal1 overnight.Then do the ligation.
My control is the vector+ligase+buffer, no insert.
After transform competent cell, I got colonies on both plates but the colonies on the ligation plate is three times as those on the control plate.
I picked ten of the colonies on the ligation plate and do the plasmid miniprep.
My problem is when I try to digest those plasmid with EcoR1, they can not be digested. it's like the EcoR1 site was lost. Even without digestion, I can see the size difference of the plasmid, some are larger than the others.
I am very confused, if the vector religated, they should be digested by EcoR1, if the insert get into vector, the plasmid also should be digested by EcoR1.
Any suggestion would be appreciated!
how did you ascertain the size is different. did you try to linearize the clones with some other enzyme?, say an enyme which is present only in the insert or only on the vector.by the first method you will know whether the insert is there.
try to check your buffers and enymes also