gel purifications of DNA fragm > 10Kb - (Aug/08/2008 )
I want to subclone a BAC clone of approx 165 Kb in subclones of 8-13Kb into a BamHI digested vector. I have to isolate fragments (8-12Kb)of a partial digest out of a LMT agarose gel. I made use of the Gelase system, so first cut out the desired gel slices, add 1x Gelase buffer for an hour at RT. Then several minutes at 70C waterbath and after that I add the gelase enzyme and incubate at 45C for an hour. After that I want to precipitate the DNA by an ethanol/ammoniumacetate precipitation. Overnight incubation at RT. After centrifugation and washing 70% ethanol I dissolve the dried pellet in 50ul TE. I checked an aliquot on gel and unfortunately couldn´t see ´hardly anything!! Who of you got experience in isolating big fragments out of an LMT agarose gel?
generally I use electrolution.
My lab doesn't have a proper eletrolution tanks so we make do using a dialysis tubing and electrophoresis tank. But if you got some cash on you, a proper electrolution tank (and assocaite kit) saves a lot of hassle.
The kit of Amersham is a good one. It is for pcr but it works, The limits of the fragments are about 12kb.
OK thanx for the replies.
Capu you mentioned the Amersham kit. Do you also have some experience with the Qiagen kit?
Best regards Jackson
Qiagen maximum is 10kb. But if you are able to accept significant loses, I have gone as high as 14kb. This is probably similar to Amersham's kit.
I've used the Qiagen kit and tried to isolate fragments in the range 7-13Kb. After isolation I could see faint bands on gel. I've used these fragments for a ligation and today I've done the transformation... Hopefully I've enough white colonies.........
Best of luck with it.
I've used Invitrogens kit with more succes than Qiagen kit for DNA fragments of about 10 kb. I did elute twice in tris and had it on there for some minutes to get yield as large as possible (though I could afford losses).
I've used the Qiagen kit to isolate the fragments 8-12Kb from gel. I've checked an aliquot on gel and there were some faint bands. With these fragments I've done a ligation and a transformation. I've got colonies however most of these were blue. I took a control with no insert and a control with the non dephosphorylated vector. In the no insert control there were (as expected) no colonies. The non dephosp vector gave a lot of (only blue) colonies (as expected).
Does anybody know why almost all colonies are blue?
poor ligation efficiency of the insert to the vector. But that is to be expected for the inserts of this size (>10kb). And depending on the size of the vector, it is also possible that transformation efficiency has already become affected.
The important thing is to use a high concentration of good quality inserts. The vector will show a tendency to ligate to smaller insert molecules, so make sure the insert is clean. And if the vector is big, the competent cells will have a tendency to become transfected by smaller plasmid molecules.
If your total plasmid size is now touching 20kb, I would recommend that you start using company purchased supercompetent cell such as One Shot genehog from invitrogen. The transformation using homegrown competent cells may start getting difficult.
Does someone of you got experience with electroelution of DNA fragments out of an agarose gel?
I've found the Elutrap Electroelution system from Whatman.
Best regards Jackson