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