User:Anthony Salvagno/Notebook/Research/2009/10/28/Redoing Unzipping: Anchor PCR failed

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Troubleshooting

  1. Run gel with 20ul
  2. Check thermocycler temperatures with thermocoupler
  3. Run PCR @ Osley Lab
  4. get more pRL574
  5. Try PCR with Tube setting (currently using Sim Tube)
  6. nanodrop primers
  7. 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

{{#widget:Google Spreadsheet |key=tuhOqwn5ymnqv4SyMQTmucg |width=1000 |height=300 }} {{#widget:Google Spreadsheet |key=tQItPDIPjA7sJm0q4VWqLAQ |width=1000 |height=300 }}

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?