Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18

Main project page Previous entry      Next entry

3/18/22 Cutting inserts to be placed in mammalian vector A) Get biobricks and enzymes from freezer. PstI, XbaI The following list of parts are: 1. BL01 2. BL05 3. BL09 4. BL12 Stock Digest B)Follow digest procedure: DNA 20.0 µL Enzyme 1 1.0 µL Enzyme 2 1.0 µL 10x buffer 3.0 µL dH2O 5.0 uL = 30.0 uL Digest Order: Biobrick Restriction Enzymes 1. BL01 XbaI/PstI 2. BL05 XbaI/PstI 3. BL09 XbaI/PstI 4 BL12 XbaI/PstI 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) 15.0 μL XbaI 1.0 μL PstI 1.0 μL Green 10x FastDigest buffer 1.0 μL dH2O 10 μL   30 μL --> 37°C/ ~10 min. # Biobrick ng/µL 1 hPCD : mCh : SP1AB (BL01) 18.834 2 fshPCD : mCh : SP1AB (BL05) 14.471 3 flyPCD : mCh : SP1A (BL09) 7.302 4 hPCD : BL01 (BL12) 10.84