User:Anthony Salvagno/Notebook/Research/2009/09/09/Ligation of pBS to SapCap

Digestion Results
I digested for about 6 hours. Then I cleaned up the DNA, but forgot to use a fresh eppi and eluted into the used collection tube. I ran a nanodrop of that DNA and got 1.2ug in 30uL. That is only half of what I put in and a real bummer. I just did another nanodrop and got 50ng/uL with 28uL left. I'm going to pretend I have 1500ng of DNA in there. I will ligate 1ug of DNA to the SapCap and save the rest for gel analysis. I feel like I'm going to need to digest again in a couple of days.

Ligation
Run for 1 hour. No changes in protocol (mix, put in thermocycler 16C, take out). I don't know why but I don't feel very confident in anything I am doing right now. I hope my calculations are good, although it shouldn't matter too much because I just loaded it with cap. Tomorrow I will clean and run in gel (.8%). {{SJK Comment|l=22:49, 9 September 2009 (EDT)|c=good notes, one important question, though: did you do ligation at 37C for real? I'm pretty sure I've never done anything above room temperature (see NEB) and I think I usually do 16C. Also it'd be good to note here what ligase and buffer you're using (e.g., "same ligase and buffer as before," or "new ligase from last week, NEB product ___"}...As a final note, I remember a lot of folklore about ATP going bad in the ligase buffer, so I always tried to treat it gently.}}}

Anthony Salvagno 10:40, 10 September 2009 (EDT):Whoops. Thanks Koch for the point out. I ran it at 16C cause that's what I have programmed, but must've blanked and wrote 37C. I changed it. Also you have some interesting notes. As far as what ligase and buffer I used, we only have 1 of each so I used the same ones as last couple of times. I feel like I treat the buffer gently. I give a finger agitation to stir it up prior to mixing, but never anything more than that. We'll see the deal today.

Steve Koch 11:00, 10 September 2009 (EDT): Whoops, my mistake: by "gently" I meant in terms of temperature. You should be able to mix the hell out of it, I'd think. Mixing is good. But until we know more, I wouldn't thaw it at room temp or with your hands. Try to thaw it on ice.