Haynes Lab:Notebook/Engineering PC-TFs/2012/12/10

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Revision as of 03:22, 14 December 2012

Engineering PC-TFs Main project page
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Summary

  • Gel Electrophoresis (PCR Primer Reactions)

Follow steps for gel electrophoresis.

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 30 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 30 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thin "teeth" and slip in the notches.
  • Waited 10-15 minutes for gel to cool. Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • The gel was poured into tray. Wait twenty minutes for gel to settle.
  • Wash the agarose gel flask.
  • Pipette 15 μL of DNA ladder into first well. Pipette 5μL of PCR reactions into subsequent wells.
  • Turn on electrophoresis (105 V) and let run for thirty minutes.



PCR of Gibson BioBricks

  • ---Karmella 13:55, 10 December 2012 (EST): Do not gel purify PCR products. Instead, Zymo DNA Clean & Concentrator kit to clean up the PCR reactions. Elute with 30 μL H2O. Measure concentration on the plate reader.
  • ---Karmella 13:55, 10 December 2012 (EST): Give me your pSB1A3 so that I can do a retransformation



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