Cloning difficulty - Problems subcloning a very large fragment... (Aug/01/2005 )

I am having difficulty cloning an 11kb fragment into a 6kb vector.

I have tried double, and sequential digests of both vector and insert followed by gel and/or column purification of my DNA. I have also tried electroporation, heat shock, lower growth temperatures following ligation with and without PEG, using rapid ligation kits, normal T4 ligase and ultracompetent cells. NO LUCK!!!

Is this kind of large cloning even possible under standard conditions?

I am extracting the fragment from a bluescript vector using KpnI and NotI and trying to ligate into a pTracer reporter vector.

Any advice would be greatly appreciated...thanks!

Oliver.

I have not much experience of clonging the large fragment.

Maybe the ratio of insert:vector is very important in this case. The optimal ligation ratio may lead to sucess. Otherwise, the liagation time may be longer than usual.

Thanks for the advice.

I have tried the following molar ratios of fragment to vector:

1:3, 1:1, 1:3

I think its a more fundamental problem with my DNA processing/purification steps but Id like to know if anyone has done such a large cloning successfully and if there are any important steps.

Maybe the vector could not accept so big insert.

i am also facing the same problem ,i want to clone in pbluescript insert are from genomic dna digestion , fragment size are varying from 6-10 kb, but every time ligation reaction failure, sometime got 3-4 clones but there are no repeatation
help please

Hi!
It is very well known that for the insert fragments of larger size the insert to vector molar ration increases. You can go upto 10:1
gud luk