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

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.

• Zymo DNA Clean & Concentrator Kit

Follow these steps for Zymo DNA Clean % Concentrator Kit

• Add 30 μL of DNA Binding Buffer to each 15 μL PCR Reaction (#1,2,3,4,5)
• Pipette up and down mixture several times to make sure it's thoroughly mixed.
• Centrifuged at top speed for 30 seconds. Flow through was discarded.
• Added 200 μL of Wash Buffer the column and centrifuged for 30 seconds. Repeated step.
• Spin Column for each PCR rxn placed in new 1.5 mL tube.
• 30μL of water was direct added to the column to elute DNA.

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