User:Anthony Salvagno/Notebook/Research/2009/11/02/Redoing Unzipping: Adapter Preplanning
I really have no clue how I should go about this. I have supposedly annealed top and bottom adapter in my box here. I have previously done this step by allowing boiled water to slowly come to RT. If I am to make my own, then I should probably just scrap what I have and do it over. I need equimolar top and bottom oligos in order to do this. I would also need to recalculate the molarities provided in our tubes via nanodrop, then do the annealing in the PCR machine. As far as settings go though, I don't know what I should do.
Start at 95C (maybe 99C) and increment temp down to 4C at some rate. What that rate is, I don't know. (Steve Koch 01:33, 3 November 2009 (EST): You'll have to read the manual to figure out how to program the ramp rate. It's a bit arcane, but I figured it out twice in my life, though I can't remember how now. I do have a program on that machine that does it.)
I will be basing work from prior measurements on this page:Oligo Annealing 4/27/09
- Top Adapter: 583.9ng/ul
- Bottom Adapter: 2750.1ng/ul
Surprisingly not too different from the April readings. So I will just go with the concentrations I have for those two oligos.
Now how should I set the PCR machine?
Koch wanted me to do some native PAGE studies with this. I will have him help me with this tomorrow because I can't remember how to run PAGE. Any comments about this are more than welcome as well.
- Steve Koch 01:36, 3 November 2009 (EST): The native PAGE is not trivial, and we may not have the correct equipment to image a good dye. So, I'd say we don't need to do this step right away. Something just occurred to me to try If I'm correct, one strand provides both overhangs, correct? If so, then we could tolerate an excess of the short (non-overhang) strand. This would ensure that all complementary overhangs were from duplexes. I haven't thought this through, very well, though, so it may be too risky and should probably stick to your best estimate of equimolar.