User:Anthony Salvagno/Notebook/Research/2009/09/17/Everything Redo (Gel, Extraction, Ligation, Gel, Extraction)
Gel
I am running a gel of the digested pBR322 to separate the two strands.
Looks like an incomplete digestion.
Nanodrop Results for Extraction
Long: 34ng/ul --> 21nM Short: 26ng/ul --> 21nM These are in the freezer labeled L and S (for long and short strand respectively). There is another tube labeled S in there that is the same stuff as this S, but one day older (maybe two I can't remember).
No Ligation today
Instead I am going to reanneal T+B, PCR a new anchor, then once I get the BstXI in I will digest the anchor with that and try ligations on Tues.
Anchor PCR
{{#widget:Google Spreadsheet |key=tIebz_3LqHQRfe7_QKliQkQ |width=800 |height=200 }} Put 90ul in 5 tubes. Run reaction like on this day.
| Stage | Temp (C) | Time (mm:ss) | Reps |
| 1 | 94 | 2:00 | 1 |
| 2 | 94 | 0:30 | |
| 60 | 1:00 | ||
| 72 | 2:00 | 35 | |
| 3 | 72 | 9:00 | 1 |
Notes
- I ran out of pRL574. I feel like I got at least 10 of the 15ul, but who knows. We'll need to transform pRL574 if we want more. I remember I saved a tiny bit for this very reason a while ago (beginning of the summer). I think it may be in my box.
- Steve Koch 23:22, 17 September 2009 (EDT):I am sure I have plasmid around somewhere. We'll have to go over this next week, but you actually only want to use a little bit of plasmid in PCR reactions, and thus it should last "forever" if you're using it for PCR. My stock I use for PCR is actually a 1:100 dilution of a miniprep. Also: I have a whole shitload of glycerol stocks of pRL574 from when Landick first sent it to me.
- Oh yea the house Taq isn't house at all but purchased. It was 5U/uL so I just used 5uL total. This is what I did over the summer for the stretching DNA.
Re-Annealing
I decided to reanneal T+B just in case that is also bad. The thermocycler is in use so I am going to improvise.
- I filled a plastic container with water
- Put it in the microwave and heat until water boils
- Put eppi in something that can float and place in the hot water
- Allow water to slowly cool to room temp (just by leaving it on counter top)
- Done!
