Problem in Restriction Digestion... - why is it cutting something it is not supposed to?? (Oct/20/2007 )
Hi alll....
I have a doubt in restriction digestion... I shall explain from the first. I have a vector wherein I can clone 2 inserts. I have cut the vector with KpnI and XhoI for one insert and cut that insert with the same enzymes. The insert is in pcDNA vector which has restriction sites for HindIII and XbaI, although these will be cleaved off since HindIII is present before KpnI and XbaI is present after XhoI. I have succeeded in cloning this insert into the vector. Now I have cut the vector for another insert using hind3 and xba1. and ligated the insert which is cut with the same enzymes (this insert is also from pcDNA vector). I also transformed them. PLease note that i dephosphorylated the vector after restriction and purified as well. I had a control which contained the restricted vector and then my ligated vector with insert DNA. I got colonies in both the plates, although the control plate had fewer colonies, giving rise to the possibiility that my ligation would have worked. So i picked colonies off my ligated colonies and carried out colony PCR which dint show a good result. so i grew them up and restricted them for my insert. The result was puzzling to me!!! it had cut out something similar to my 1st insert which i had succeeded in cloning earlier on.As i mentioned, it is not supposed to cut out this insert since it does not have the sites for hind3 and xba1. it is also not cutting out my secong insert since the size is atleast 600bps less and its not the vector it is cutting out, since hind3 and xba are just almost 100bps apart in the vector.
Can anyone tell me what maybe happening here? Thanks....
I have a doubt in restriction digestion... I shall explain from the first. I have a vector wherein I can clone 2 inserts. I have cut the vector with KpnI and XhoI for one insert and cut that insert with the same enzymes. The insert is in pcDNA vector which has restriction sites for HindIII and XbaI, although these will be cleaved off since HindIII is present before KpnI and XbaI is present after XhoI. I have succeeded in cloning this insert into the vector. Now I have cut the vector for another insert using hind3 and xba1. and ligated the insert which is cut with the same enzymes (this insert is also from pcDNA vector). I also transformed them. PLease note that i dephosphorylated the vector after restriction and purified as well. I had a control which contained the restricted vector and then my ligated vector with insert DNA. I got colonies in both the plates, although the control plate had fewer colonies, giving rise to the possibiility that my ligation would have worked. So i picked colonies off my ligated colonies and carried out colony PCR which dint show a good result. so i grew them up and restricted them for my insert. The result was puzzling to me!!! it had cut out something similar to my 1st insert which i had succeeded in cloning earlier on.As i mentioned, it is not supposed to cut out this insert since it does not have the sites for hind3 and xba1. it is also not cutting out my secong insert since the size is atleast 600bps less and its not the vector it is cutting out, since hind3 and xba are just almost 100bps apart in the vector.
Can anyone tell me what maybe happening here? Thanks....
it sounds like your vectors has all four sites, and your insert has all four sites. If you clone the insert with the internal sites into the vector, that construct will still have all four sites (two that you used, and two flanking that were on the vector.)
Otherwise, I'd suspect contamination.