Electroporation into ES cells
互联网
1052
Electroporation DNA Preparation: 1. Cut the required amount of DNA (banded on a CsCl gradient) with the appropriate restriction enzyme and check for complete digestion by running 500 ng on a minigel. The DNA concentration should be no higher than 1 ug/ul in the large-scale digest. 2. Extract the large-scale digest once with an equal volume of phenol/chloroform and once with an equal volume of chloroform. Precipitate the DNA with 2.4 volumes of ethanol, spin down and dry in the Speed-Vac. 3. Resuspend the DNA at the desired concentration (usually 1 ug/ul) in sterile 0.1X TE (25 ul of DNA per electroporation). Measure the DNA concentration using the fluorometer. Cell Preparation: 1. Embryonic stem cells (80% confluent) should be passaged 1:2 the day before electroporation. Cells to be electroporated should be fed 4 hours before harvesting. 2. Trypsinize cells and resuspend in media (cells from 2 x 10 cm plates can be combined in a total volume of 10 ml in a 15 ml tube). 3. Pellet the cells and aspirate off the supernatant. Resuspend in 10 ml PBS and determine the total number of cells by counting a 20 ul aliquot. (Note: The usual yield is 30 x 106 cells per 10 cm plate.) 4. Pellet the cells and aspirate off the supernatant. Resuspend in PBS at a density of 11 x 106 cells/ml. Count a 20 ul aliquot to confirm. Electroporation: 1. Add appropriate amounts of DNA and cells together in a 15 ml tube (25 ul of DNA and 0.9 ml of cells for each electroporation). 2. Allow to sit at room temperature for 5 minutes (this step may be omitted). 3. Aliquot the cell/DNA mixture into electroporation cuvettes (0.9 ml per cuvette; the volume is important!). Place the cuvette in the electroporation holder with the foil electrodes in contact with the metal holding clips. 4. Set the Biorad GenePulser at 230V, 500 uF (requires the capacitance extender) and press the two red buttons to electroporate. The machine will flash "Ch 9" and will beep when electroporation is complete. (Time constant should read between 5.6 and 7.0.) 5. Leave the cuvette at room temperature for 5 minutes and then plate the cells at an appropriate density (up to 2 x 107 cells/100 mm plate or 6 x 106 cells/60 mm plate. DO NOT EXCEED THIS DENSITY, since G418 takes 3-4 days before killing starts and plates will become over-confluent.). If G418 selection is to be applied, this will be done 24 hours post-electroporation. (NOTE: G418 concentration must be titrated for every batch.) 6. Refeed the plate(s) with fresh media + G418 every day for the first 6-7 days (until colonies are visible and most cell debris has been removed). If using FIAU (0.2 uM) selection, this may proceed simultaneously. 7. The typical yield for RV4.0 is up to 104 colonies/107 cells/100 mm plate. Other constructs will deviate significantly (and unpredictably) from this yield. 8. Colonies may be picked as early as 8 days, are best around 10-11 days, but may be recovered up to 18-21 days after the electroporation. From the Laboratory of Dr. Allan Bradley Baylor College of Medicine,Houston,Texas