AvrII??? Friend or Foe? - molecular cloning; AvrII; digestion; resistance (Jul/08/2010 )
Long time lurker first time asker.
I wanted to know about AvrII. I've been cloning an 8 Kb fragment into a 6 Kb vector using KpnI (HF variety, NEB) and AvrII (NEB).
I've sequenced the vector and I've verified the presence of both KpnI and AvrII
Both I perform single digests and KpnI cuts well.
AvrII fails to cut.
Does anyone have tricks, tips or brand suggestions so as to get a digestion at my AvrII site?
1) Check that the enzyme works... get another sequence with an AvrII site and cut that.
2) Check the buffers, if AvrII requires a special buffer (e.g. contains ATP or something else degradable) it may require aliquotting to stop it degrading with defrosting
3) Check incubation times/proceedure and special conditions required on other suppliers and have a go with them, they should work as well (odd temperatures, long/short incubations, degradation of enzyme over the time, amounts of DNA it can cut...).
Is AvrII methylation sensitive? if so, the plasmid may be methylated at the restriction site, which will mean that the RE won't cut. There is very little you can do about this.