ligating small inert into a large vector
Posted 12 January 2011 - 10:39 PM
Posted 13 January 2011 - 03:27 AM
Posted 13 January 2011 - 04:59 PM
Do you have any controls if the double restriction was OK? Did you see the cleaved products? In my experience a low transformation yield is usually due to poor restriction. I would not suspect EcoRI because (at least in my hands) it is very robust, but for KpnI I honestly don't know. How did you generate the 125 bp product? Did you leave enough space for the restrictase to cleave?
I did not use any controls to check double restriction digestion but I have checked the efficiency of restrictions enzymes individually on the same plasmid. Digestion efficiency seems to be fine. The 125 bp product was generated by annealing two long primers of 70 bp and extension of their overhang ends at the 3' (Pardon me for i said PCR before, infact this was just extension of primers). I have checked this product on the gel for its size. This 125 bp product possesses those restriction sites at its ends and I have made sure there is enough space (4 bases before the restriction sites) for the restrictase to cleave.
Edited by laborat, 13 January 2011 - 05:09 PM.
Posted 13 January 2011 - 08:57 PM
Posted 13 January 2011 - 10:45 PM
just to double check. Do the inserts overhangs complement the overhangs on the vector? KpnI leaves a 3' overhang while EcoRI leaves a 5' overhang
the vector is also digested by the same enzymes. and the overhangs complement