Haynes Lab:Notebook/Engineering PC-TFs/2013/09/12

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September 12, 2013 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 45 min at 100 V. Check gel under UV light. Reagent Volume 30 μL/lane, 1% agarose; Ladder DNA (plasmid) 20.0 μL SpeI 1.0 μL PstI 1.0 μL Green 10x FastDigest buffer 3.0 μL dH2O 5 μL   30 μL --> 37°C/ ~10 min. ZymocleanTM Gel DNA Recovery Kit Add 3 volumes of ADB Buffer to each volume of gel (600 μL). Incubate at 55°C for 10 minutes. Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through. Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step. Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH2O to elute DNA # Sequence ng/µL 1 CMV -1.17 2 CMV 2.767 3 CMV -2.116 5 MV9 20.838