User:Anthony Salvagno/Notebook/Research/2009/09/17/Everything Redo (Gel, Extraction, Ligation, Gel, Extraction)

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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

View/Edit Spreadsheet

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.

  1. I filled a plastic container with water
  2. Put it in the microwave and heat until water boils
  3. Put eppi in something that can float and place in the hot water
  4. Allow water to slowly cool to room temp (just by leaving it on counter top)
  5. Done!
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