E/P Digest for FURI 2013-2014 application
- Retrieve BL01, BL05, BL09, BL011 assembly from freezer and thaw at room temperature.
- Also let restriction enzymes thaw.
- Do restriction digest (EcoRI/PstI)Diagnostic digest
||15 µl Total
| ||1 rxn||x 4
| DNA plasmid||3||--
| enzyme 1||1||4
| enzyme 2||1||4
| 10x buffer||1.5||6
- Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
- Place each tube in heat block 37°C.
Follow steps for gel electrophoresis.
- Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
- Fill gel flask with up to 60 ml of TA buffer.
- Create 1% gel by putting .6 grams of agarose into flask.
- Microwave agarose solution for 40 seconds
- Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 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 thick "teeth" and slip in the notches.
- Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
- Pour gel into tray.
- Wash the agarose gel flask.
- Run gel for 30 min at 100 V. Check gel under UV light.
- 2 mL LB Broth + amp added to test tubes
- Use pipette tips and dip in bacteria colony.
- Performed Zyppy Miniprep Kit