Dear all!
During a ligation something occured which I can’t interpret yet.
I used the cloning vector pPCRScript(Cam) containing a part of a new
target gene flanked by the restriction sites BamHI (5’) and XmaIII(3’).
The expression vector (pET20b) with the original gene was also cut with
BamHI and XmaIII, respectively. Both, the new target gene and the rest
vector were gel eluted. Then ligation was performed using T4-DNA-Ligase.
After transformation several clones were checked on mini expression
level resulting in only few proteins with the right size and many which
were 15 kDa smaller. Sequencing information confirmed that the
expression vector for the smaller protein contains a stop codon due to
the loss of 28 nucleotides from the 5’-end of the insert. On the other
hand, the vector expressing the right protein misses 45 nucleotides
after the terminal XmaIII restriction site, suggesting that those
nucleotides where cut from the original vector during the ligation
(fortunately that doesn’t harm us).
Did anybody experience something similar?
I read something about lyase activity of ligases. May this be the
reason?
Thank you for your help.
Kai
