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
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