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Tuesday, Day 7
Electroporation: Use three of the 10 cm dishes of inactivated MEF cells prepared on Day 6. From each dish, remove the old media and add 10 ml of fresh ES media. Place these dishes back in the incubator. Next, take the 10 cm dish containing the MEFs and ES cells and remove the old media. Rinse the dish x 1 with PBS, then add 2 ml Trypsin/EDTA and incubate for 5-10 minutes at 37oC. After 5 minutes, the cells will look like "bunches of grapes" under the inverted scope. Add 2 ml more Trypsin/EDTA to the dish, pipette up and down to break up the clumps and incubate for 3 more minutes. [You must have single cells for the electroporation.] Look at the cells again under the inverted microscope. The MEFs are the larger cells, and the ES cells are small and shiny; most if not all should now be single cells. To the 10 cm dish add 7 ml ES media, pipette up and down, and transfer all the cells to a 15 ml centrifuge tube. Pellet the cells by centrifuging gently (1000 RPM in a Sorvall tabletop) for 5 minutes at 10oC. Take off the supernatant and resuspend the pellet in 1.0 ml ice cold 1x Hebs (see Reagents, Transfection Buffer 1 X Hebs). Prepare a 5 ml tube of ES media for the cells after electroporation. Get out a sterile "flat pack" 1.8 mm gap cuvette (BTX order #485) and insert the cuvette between the safety stand contacts. Make sure there is a good contact between the cuvette and the safety stand contact. Having the safety stand connected to the rear of the unit using the cables supplied, turn on the power switch. Set electroporator (BTX 600 or equivalent) as follows: 500V/Capacitance and resistance, 500uF capacitance timing, 360 ohms R8 Resistance timing, Charging voltage 185V. Pipette the ES cells up and down with a 5 ml pipette and add to a microfuge tube containing the targeting construct DNA ( 40 ug of clean linear DNA in 1 X TE @ 1 ug/ul for each electroporation). Pipette cells and construct up and down with a pasteur pipette carefully. Slowly add the cells to the cuvette, taking care not to introduce any bubbles. Slide the cuvette into the electroporation chamber, dial the charging voltage to 185V and push the pulse button. Wait until the charging is over, then push the reset button, dial down the voltage, and turn the power off. With a sterile pasteur pipette, take the electroporated cells out of the cuvette and place them into the 5.0 ml of fresh ES media in a centrifuge tube (final vol. = 6 ml total). Take the three 10 cm dishes of inactivated MEFs (freshly fed with ES media) and add 2 ml of the transfected ES cells per dish. Label the dishes with the targeting construct's name and date. Rock the dishes slowly to evenly disperse cells.
Alternate Electroporation Procedure using Safety Chamber 630 A, BTX cuvette, 2 mm gap:
Prepare a 5.2 ml. tube of ES media for cells after the electroporation. Prepare ES cells for electroporation as described in original text on Day 7, except add only 800 ul cold 1 X Hebs to your pelleted cells. Place a sterile BTX cuvette (2 mm gap) in a 630 A Safety Chamber and attach the electrodes securely to the Electro Cell Manipulator 600. Pipette the cells up and down with a 5 ml pipette, and add 800 ul of cells to a microfuge tube containing the targeting construct (40ug of clean linear DNA in 1 X TE @ 1 ug/ul for each electroporation). Mix the cells and DNA with a 200-1000 ul barrier tip trying not to create bubbles. Add 400 ul of the cell/DNA mixture to a BTX cuvette. Set Electroporator as follows:
500Vcapacitance and resistance, 500uF capacitance timing, 360 ohms R8 Resistance timing, Charging Voltage 160V. When capacitors are charged hit the pulse button. When charging is complete, with the pipette provided, harvest the electroporated cells and place them into 5.2 ml of fresh ES media. Repeat the electroporation with the other 400 ul cells/construct in a new BTX cuvette. Add this to the 5.2 ml yielding a final volume of 6.0 ml. Take the 3-10 cm. dishes containing inactivated MEFs freshly fed with ES media and add 2 ml. of the electroporated cells per dish. Rock the dishes slowly to evenly disperse the cells. Label the dishes and place in the incubator.
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