large fragment cloning - (Aug/05/2004 )
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!
I do not have experience with pBlueScript, but have successfully done cloning of 6.5 kb into a 7.6 kb vector. I had more luck with electroporation than with heat shock. For electroporation, the amount of DNA used is highly critical. In my case, >2ng of vector construct (I ethanol ppt before using it for electroporation) gave few or no colonies. I used 'home-made' electrocompetent DH5a. Condition used, 200 ohms, 25 capacitance and 1.5 kV. Hope that helps.
I had used the pBluescript vector to construct a plasmid, the size of which is more than 20 Kb. the competent cells which I use are JM109 and XL-1 blue, normal transformation is ok.
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.