User:Anthony Salvagno/Notebook/Research/2009/09/14/SapI and EarI Notes
Requires 1unit and 1 hour of digestion. Do not do more than one hour. Possibly include more enzyme. The FAQ page on this enzyme is hilarious. They use a bunch of one line answers like they don't really care to speak on the subject. I used similar technique in 9th grade when we were required to write complete sentences even if I could suffice with a one word answer. Interesting quote (from NEB.com): Q1: Why isn't SapI cutting?
A1: Unlike most other enzymes, SapI tends to settle in the tube. Mix with your pipette prior to removing from tube to ensure digestion.
I feel like I knew this... let me prove it. I remember back in the day this particular gel image. I remember Kelly and I discussing the results and I'm pretty sure I said something to the effects of "I think the enzyme settled in the tube." I actually remember "seeing" the enzyme settle and not worrying about it. I say "seeing" because when one mixes RE or DNA in solution you can see it go in I guess because of differing buffers. I always feel nervous about my experiment when I don't see this happen.
Good for extended digestions (overnight >8hrs). In the event of long digestion, consider using smaller enzyme amounts ie .25units. It seems no real problems exist.
Lucky for us the EarI site is the same in pBS as it is in pBR322 so we are going to work out the method with pBR322 and then try our hand at pBS when I get more time and can do transformations. We can ligate to the same unzipping construct. The idea this time is that I will gel extract a large fragment (3 cut sites) and ligate that to the UC. This piece in pBS will have the MCS which contains our insert. Although I just realized I have to be leary of cut sites within the insert for EarI.
I also want to reanneal the SapCap because it wasn't working right and I want to make sure it is not doing some funny business.
- Can I literally take the tube of dsCaps and reheat and anneal? (Steve Koch 23:12, 14 September 2009 (EDT):Yes!)
- I need to make new annealing buffer. I should have used 500mM NaCl instead of 50mM cause I made a 10x stock. Also need to make more 4M NaCl.
- I would like to put some SapCap in ligation rxn to see if that is the problem or if that the pBS is weird like that.