Experience with NheI, AsiSI, FseI and MluI? - (Oct/12/2005 )
I want to ligate some inserts with some uncommun Enzymes. The REs I want to use are 1: AsiSI and MluI and 2: FseI and NheI. Does someone has experiences with those Enzymes? Do they need any special treatments? I already know that AsiSI cannot digest supercoiled DNA
and has star activity and doesn´t like to be stored at -70°C, so what about the others?
I have used Mlu I and Nhe I many times without issue...
Nhe I never give me problems...
MluI can not digest if CpG methylation occurs. So do Fse I and Asi S I.
But for Fse I, -70° storage is recommended for sotrage more than 30 days.
Neb catalog tells about star activity only for AsiS I.
For star activity, i use 2h digest, and if it's ovenight, i use a PCR machine to low the temp after 2h.
Hope tat helps
Check out the new england biolabs catalog if you can find one lying around, There is really good information in the appendixes about the activity of different retsriction enzymes, eg. methylation sensitivity, buffer composition, ect.
I already looked in the NEB Catalog, but for example, that AsiSI can´t digest supercoiled DNA wasn´t one of the informations in the Catalog. I found it out by accident. Then I called NEB service and the asked. That was when they confirmed my information about AsiSI.
I thougt that I could save a lot of time, effort and frustration, in case someone has similar experiences with one of the other RE´s. That´s that.
I starting today, wish me luck, that the digestions and ligations are going to work