Haynes Lab:Notebook/Engineering PC-TFs/2013/03/03

Summary

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

• 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.

• Label Tubes

1. BL01 2. BL05 3. BL09

• 2 mL LB Broth + amp added to test tubes
• Use pipette tips and dip in bacteria colony.