blunt-cloning - all are vector self-ligation products? (Jul/11/2003 )
Add some DraI in your ligation reaction. Relegated vector (without insert) will be recuted. Constructs with insert will not be affected.
Hi! I´m having problems with blunt end ligation too and i´m getting a little bit desperate by now...I´don´t know what else to try so I hope some of you can help me. The thing goes like this:
I need to clone a Tet resistance gene inside a haemolysin gene which I have already cloned into a vector (this I did by using the TOPO kit). I got my vector by PCR with Pfx from Promega by using internal primers to the haemolysin. This means that, supposedly, my vector has no overhangs (completely blunt ended) and no 5´phosphates. I purified this vector from the gel by using a gene clean kit. I got the tet R gene by digesting a plasmid with Sma I and purified it by gene clean as well. Later I mixed the insert and the vector in a 8:1 ratio, transformed E coli TOP 10 competent cells, plated them onto a LB+tet agar and nothing happened...not even a single colony. I did this like a million times trying to change the DNAs, repurify6ing things, reconcentrating them and still nothing...what is it that I´m doing wrong?
Thanks in advance
In my practice, I never had such problems with blunt end ligation.
I normally use a quick ligation kit from Roche and I keep my ligation reaction for 1 hour at 15 degrees. Never did dephosphorylation. Yet I do find vector self ligations (3 or 5 out of 10) but its ok. and whenever I do dephosphorylation, it surely decreases the ligation efficiency. Furthermore, I almost always keep the vector to insert ration of 1 to 10. and dont make your ligation reaction go beyond 10-20 microliters.
what's the different between PEG4000 and PEG8000? I found that in the ligation buffer of Invitrogen, they included PEG8000 at 25%. So, can the addition of PEG4000 into ligation mixture improve the efficiency?