Ligation problem, religation of backbone without any insert?
Posted 13 October 2010 - 03:13 PM
Insert for cloning was generated by PCR and has been checked on gel for correct size. After the restriction sites there are 6 bp added to each end to make sure the insert is digested. Target vector has been digested by both enzymes cutting out a 1200 bp old insert and the 7300 bp backbone. This was checked on gel and looks like ~100% digest.
Ligation was performed by cycling ligation (8 hours of 30 second 10 degree and 30 seconds 30 degree) with 10 minutes 70 degree heat inactivation. I've ligated 50ng vector backbone with 250ng insert or with just water.
And the result ... way more clones on the plate with insert than without. Checking clones by PCR with three (!) different primer pairs: the distance covered by the primer pairs is 1430 bp, 1550 bp, 1650 bp with all of them used before and working fine. The later one is binding outside the insert on both sides, the other two pairs bind each inside the new insert and outside on the backbone.
Puzzling result is each PCR gives around 100 bp on each clone - except a religated one that still has the old insert with the correct size (around 800 bp)...
I guess now (minipreps are under way to digest these clones) that ... the ~100 bp thing is probably just dimer because there is a re-ligation of the vector backbone without insert. I just don't get why than the primer pair which binds on each side outside of the insert doesn't give a band ... The backbone has 4 bp overhangs and they are not compatible at all.
So in case the ligation truly is the problem here how can I optimize it? The main problem I suppose to be effective here is that there is pre-microRNA structure inside the insert and some years back when I cloned the whole cassette into the backbone it took me several month before I got a single positive clone ... But there simply is no other restriction site available I could use to avoid having to ligate that secondary structure nightmare again.
Anyone knows about ligation protocols that help prevent the favoring of re-ligation of incompatible ends because of secondary structures in the insert?
Posted 15 October 2010 - 02:16 AM
Posted 20 October 2010 - 01:22 AM
Your new insert is around 1600 bp .. correct? You say you use 250 ng insert and 50 ng backbone. If your insert indeed is 1600bp, then your fragment:vector ratio is too large (25, whereas 3 is adviced). Also, I don't know how much you transform but I would stick to a total of 100ng DNA, because more won't increase the number of clones that much.
The backbone could re-ligate, in which case you should pick up a small band with the primer pair on each side of the insert. The RE results on the mini-preps will shed more light on this issue hopefully.