Get your 2007-2008 NEB catalog
A co-worker asked if I’d help her with some cloning. She had the strategy all figured out. The oligos were here and the pcr was done for the insert. All I had to do was digest the insert fragment and the vector backbone, purify them and ligate them together. Seemed like it should be easy enough.
That was until I looked at the enzymes. Urgh! They were two of those enzymes you only use under dire conditions. One cuts at 37 degrees, the other at 50. AscI only cuts 100% in NEB buffer 4, the other one only in buffer 3. I gave her a hard time about passing the more *difficult* cloning on to me. She said she knew about the temperature difference but that both enzymes cut in buffer 4. Huh? I had just checked the NEB catalog and knew that wasn’t right. BsaI only says it has 50% activity in buffer 4.
What was going on? We both felt like were going crazy. She brings her 2005-2006 NEB catalog over and shows me. BsaI and AscI both cut in buffer 4 - 100%. I look in my 2007-2008 NEB catalog and BsaI only cuts 50% in buffer 4. Turns out were both right. Sorta.
I’m not sure what changed. Is NEB purifying the enzyme differently? Did they just realize it didn’t cut as well as they had thought? Whatever the reason, the recommend buffer has changed. I’m not sure how many other buffers have changed but if you have an old NEB catalog hanging around you might what to get a newer one!
PS - Turns out neither one of us has had a whole lot of success with the cloning so far. I’m still not sure which is the best buffer to use!
Posted on Thursday, March 20th, 2008 at 10:57 am Categorized as:Catalogs, Suppliers You can skip to the end and leave a response. Pinging is currently not allowed.
