PROTOCOLS\

32DELECT

10/27/98

TRANSFECTION OF 32-D CELLS BY ELECTROPORATION

A. Cells: You need 0.5 x 10⁷ cells to electroporate a single DNA.

B. DNA: You need to use 30-100 mg of gene-containing DNA per electroporation, and 1/10 that amount of selectable marker DNA per transfection (if applicable). Make sure the total DNA volume is less than 20 ml in water or 0.1x TE (sterile). DNA should be CsCl-purified or QIAGEN prepped and re-ethanol precipitated to remove CsCl and sterilize, since the presence of excess CsCl will alter the electrical parameters of the electroporation.

C. Each electroporated cell line/selectable marker deserves a no marker control to ensure that the cell is killed by the selection.

DAY 1:

CELL PREPARATION.

1) Take requisite number of cells and spin down in 50 ml conical tubes (1500 rpm, 5 minutes in table-top). Wash once in PBSucrose, and resuspend in PBSucrose at 1.5 x 10⁷ cells/ml.

ZAPPING:

1) In a sterile Bio-Rad "Gene Pulser" cuvette, add 0.3 ml of cells/PBS and 20 ml DNA solution. Cap the pulser cuvette and pulse in Montminy apparatus, using: R2, 300 V, .

2) Transfer pulsed cells into a 10 cm dish containing 10 mls of RPMI+FBS+WEHI+P/S (FW) (non-selective media) and transfer to incubator overnight.

DAY 2:

SPLIT CELLS INTO SELECTIVE MEDIUM (Best done AM of second day):

1) Remove all electroporated cells/medium from dish and place into sterile 50 ml conical tube. Add 10 mls of fresh selective medium to dish and return to incubator.

2) Spin downs cells, resuspend in 30 mls selective medium and distribute 1 ml of cells/medium to each well of a 24 well dish. Add a further 18 mls of selective medium to the cells and distribute to a second 24 well dish.

SELECTIVE MEDIA:

HISTIDINOL: 5 mM. NEO: 750 mg/ml geneticin. HAT: 80 mM mycophenolic acid+HAT.

SCALE UP AND ANALYSE CELL LINES:.

1) Check mass cultures 1 week post selection. Check wells about 10 days post selection. You are looking for live cell: they are shiny, large and assymetrically shaped. There should also be a bunch of them in the same area due to cell division. The easiest way to screen wells is to wait a little longer and look underneath the plates for fuzzy looking spots which are cells proliferating.

2) When wells get to be confluent (or close to it), split them up into 75 cm² flasks with 10 ml of media. When getting close to confluent, add an additional 10 ml. When confluent, remove 15 ml of cells and spin down. Lyse in 200 ul of PIK lysis buffer by resuspending with a pipettor. Transfer to eppie and after 5-10 minutes spin out nuclei and lordt (Danish for shit). Add LSB to sup in a fresh eppie, boil, and load to minigel. Run minigel, transfer, and immunoblot.