Hi all,

1) Has anyone used smaI as a RE to cut a vector for blunt end cloning?

Background: Was surfing the web for advise, came upon this website:
https://info.med.yale.edu/mbb/koelle/protoc...subcloning.html
Was pretty useful, but in the last sentence, he caution about smaI's activity regarding cloning.
So my situation now is I'm trying to cut a plasmid 2862bp with smaI.
I over digest overnight at room temp. Ran the whole product on a 1% agarose, gel purified using QiaQuick gel extraction kit.
omited the CIAP step as I assume that the vector is fully cut since I extracted the single band plasmid DNA from the gel.

currently doing some polishing of the DNA insert with klenow fragment...

2) So do you guys treat the smaI cut vector with CIAP, t4 DNA polymerase or Klenow fragments to blunt it?


Hope you guys can shed some light....

thanks

I always treat digested vector with CIAP or, better (in my hands), Shrimp Alkaline Phospatase...
For successful ligation, the insert DNA then has to be phosphorilated, of course, either by usage of phosphorilated Oligos in PCR or by treatment with Polynuceotide Kinase+ATP.

mike

SmaI is not the best RE out there, but I understand that sometimes you'd have to use it since it's your only choice. I'd definately phosphatase my plasmid in order to prevent self ligation. I recently used Antarctic Phosphatase and it workes also very good. Make sure that the ends of your insert have P's by Kinasing them in presence of ATP!!

Good luck!!

Sma I has an isoschizomer which may be a better enzyme. If you still need the blunt end, you can just do a fill-in or chew back the overhang.