Cloning large fragment - Cloning (May/01/2007 )

Hi-

I am trying to clone a very large fragment (7.5kbp). I have tried to PCR the fragment and by using restriction enzyme sites ligate the fragment into my vector. I have used several types of ligase enzymes and several different protocols. I then went to TOPO cloning. When doing TOPO i cloned 3 other fragments at the same time and they all worked, this one did not.

Right now i am thinking of either:

1. trying to clone the fragment into a simple vector such as pBlueScript.. then ligate it into my vector (pEF1).
2. Trying a two step cloning (ligate half into my vector, cut and ligate the second half)

Assuming that I am doing everything correctly.. does anyone have a suggestion for cloning a large fragment into a plasmid??

what kind of cells are you transforming your ligated plasmid into? And how many colonies do you screne when looking for the plasmid?

I have found that you need the extra competency of company purchased cells to get pass this hump, especially if your vector is large (10kb >) to begin with.

Do you do a kill digest to kill unligated vector? Is that possible with your current ligation strategy?

How concentrated is your insert? I guess not very, considering it is a PCR product. Anyway, I would prefer strategy 1. Since you already have the product, and you don't need to buy new primers to amplify your large products in two parts.

Finally as for the TOPO cloning, did you treat your PCR product with Taq and dATP? Proof reading polymerase almost only produce blunt end products, there is a need to put an A overhang.

Did you have any problems with making such a large fragments with PCR?

Do not have any good answers unfortunately.