large fragment cloning
Posted 05 August 2004 - 02:28 AM
I have a 9kb fragment already cloned into bluescript, and I am trying to insert an extra 3.5kb. I've been trying for some time now with no success - when I do get colonies very few have any of the new insert, and none of them all of it.
Can pBlueScript realistically take inserts of 12.5kb or would I be better off trying a different vector e.g. a phagemid or cosmid?
Does anybody have any tips for dealing with large inserts generally? I use XL-10 gold or XL-1 blue cells for the transformation, are these the best? I've heard that DH10B is good for large inserts, is it worth buying some of these cells? Is electroporation better than heat shocking (which I do at the moment)?
any help much appreciated!
Posted 05 August 2004 - 04:13 PM
Posted 06 August 2004 - 01:20 AM
Inserting a small fragment into a big plasmid is very difficult, even if you use two-enzyme digested fragment. for the small fragment has a tendency to self-ligation, this makes the concentration of the small fragment decrease very quickly in the ligation system. in order to prevent this, I dephospharylated the small fragment firstly, then performed the ligation. I had used this method for two times, all were succeeded. but in the control ligation in which the small fragment without dephospharylation, it was very difficult to pick a clone with new insert.
Edited by xysun, 08 August 2004 - 10:28 PM.