User:Anthony Salvagno/Notebook/Research/2009/10/28/Redoing Unzipping: Anchor PCR failed
Troubleshooting
- Run gel with 20ul
- Check thermocycler temperatures with thermocoupler
- Run PCR @ Osley Lab
- get more pRL574
- Try PCR with Tube setting (currently using Sim Tube)
- nanodrop primers
- Try PCR with other primers
- Do with 2.5uM MgCl2
Of this list I will do numbers 1 and 7 immediately. Temp check isn't feasible with PCR going again so I will hold off for another day. Getting more pRL574 isn't doable here so I will have to head over to Osley lab to do that, again for another day. The nanodrop wasn't working so I will have to have Brigette check on that.
PCR Redo
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Gel Rerun
So this time I did 16ul of PCR product and 4ul of loading dye. I ran the gel for about 20 min at 160V. Decent separation and no melting. The results are in:
Still no product. I am officially declaring this a failed PCR reaction. I didn't take a picture because frankly how many times can I show an image of a ladder? I don't wanna show no mo'! Anyways that's it to report. Hopefully the new PCR works. Gel to come tomorrow (unless it too has failed).
Primers Nanodropped
{{#widget:Google Spreadsheet |key=tUJNwe9cSlARl1VVkLtcBjw |width=300 |height=200 }} I don't have the specs on the primers so I can't calculate what the molarity is. Also the 1:100 pRL574 is a lower concentration than I expected. Perhaps it could be a case of I grabbed the 1ul in the tube that has less concentration. Who knows?
