Haynes Lab:Notebook/Engineering PC-TFs/2013/10/05

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October 5, 2013 PEG/MgCl2 Make 50 mL Put water, then MgCl2, then PEG 8000 in 50 mL conical tube Reagent Volume PEG 8000 15 g/mL MgCl2 1.5 mL H2O 33.5 mL Total 50 mL Perform restriction digest for CMV promoter Reagent Volume DNA (plasmid) 10 μL XbaI 1.0 μL PstI 1.0 μL 10x FastDigest buffer 3μL dH2O 15 μL   30 μL --> 37°C/ ~10 min. PEG/MgCl2 Precipitation This procedure was adapated from Size selective DNA precipitation Measure the following into 1.5 mL tubes. Reagent 15% 10% 7% 5% 2% 30% PEG 50 μL 33 μL 23 μL 17 μL 7 μL DNA 15μL 15 μL 15 μL 15 μL 15 μL dH2O 35 μL 52 μL 62 μL 68 μL 78 μL Total 100 μL 100 μL 100 μL 100 μL 100 μL Centrifuge for 15 minutes at 10,000g. DNA Clean & Concentrator Although there was no pellet, ran a DNA Clean & Concentrator on samples. Add 2 volumes of DNA Binder Buffer to each DNA sample (200 μL). Place mixture in spin column into 2 ml collection tube. Centrifuge at full speed for 30 seconds. Add 20 μL of DNA Wash Buffer and centrifuge for 30 seconds. Repeat this step. Place Zymo-Spin Column into a new 1.5 ml tube. Add 20 μL of water directly to column matrix and spin to elute DNA. Gel Electrophoresis 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. Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA, according to Lis and Schleif 1975, NAR. I kept DNA from the supernatant and discarded the precipitated DNA. Therefore, I should see larger bands at 2% PEG and smaller bands at 15% PEG.