User:Anthony Salvagno/Notebook/Research/2009/09/23/PCR That I didn't get to do yesterday

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Anchor PCR

{{#widget:Google Spreadsheet |key=tBrtxBTMYBu-_prtru4hQ7Q |width=800 |height=200 }} Put 90ul in 5 tubes. Run reaction like on this day. Also this time I will use 1:100 pRL574. Also ran out of Taq. I'm feeling like I got 3.5ul instead of the prescribed 5.2ul.

Cycler Settings

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

Gel Analysis

I realized today that yesterday's throwing out wasn't so great. I forgot that I had only put in 1ul of DNA from a 30ul stock. So when I said I had 132ng it was really something like 132 x 25 which is some larger number. I don't think it is too bad that I got rid of it because it wouldn't have been that much overall. Today if this works out will yield a ton of DNA. Let's hope and hope and hope.

Ok I ran a nanodrop and got 78.4ng/ul of DNA. But I did this because there was nothing on the gel. I don't know what to make of this. Another failed PCR reaction? I can't handle this. What can I do better? Should I change my cycler times? Maybe there wasn't enough Taq? I have no clue. I'm going to go home and think about this. I could run a gel, but now I risk running low on DNA before I even get to the ligation part. Also now I can't find the google doc of my dna concentration estimator that Koch gave!

Also of note I can't redo this ligation until more Taq comes in, which will be tomorrow (hopefully).

Update: The Google Docs magically fixed themselves and the dna concentration file reappeared. Awesome.