Protocol S.4

Stable Lines

This protocol is optimized for erythroid cells, other cell types may require modifications.

 

Solutions

G418 Stock

1 g G418 from Gibco-BRL

add Q to bring it up to 100mg/ml active drug

(this is batch specific i.e. 546 mg active/1g powder)

sterile filter through 0.22mM filter unit into sterile eppendorf

tubes, freeze and store at -20°C

Poly-L Lysine

use a high molecular weight poly-L Lysine from Sigma

reconstitute at10 mg/ml in PBS

store at 4°C

 

Procedure

poly-l lysine treat plates

• Add enough Poly-L Lysine stock to cover bottom of plate and incubate at room temperature for 30 minutes. Aspirate the poly-L-Lysine and wash with sterile Q. UV treat for 15 minutes and store in the original plastic sleeve.

prepare dna for transfection

• Linearize 20 mg of the expression construct and 2 mg of pSV2neo (EcoRI). Phenol/chloroform extract and EtOH precipitate. Resuspend the expression construct DNA in 20 ml sterile TE and the pSV2neo to 0.5 mg/ml.

prepare cells for transfection

• Spin down cells (erythroid) at 1.2K for 10 minutes (2x107 cells per construct) and resuspend in 800 ml medium per construct.

• Mix the following: 800 ml cells, 20 ml expression construct, and 4 ml pSV2neo construct and place in an electroporation cuvette on ice.

• Electroporate at 960 mF, 280 Volts (0.28 kV) and hold on ice for 10 minutes.

• Add 10 ml medium and plate.

• After 48 hours add G418 to 800 mg/ml, dilute 10 fold and 100 fold.

• Add 0.5 ml of each dilution to 16 wells each of a 48 well plate pre-treated with poly-L Lysine.

• Change medium every 2 days with 800 mg/ml G418 until cells grow out. During the first 4 days the cells look OK, then most of them die, and finally colonies grow out.

• As cell lines grow out, transfer to larger plates and reduce G418 to 400 mg/ml.

freeze down cell lines

• Spin down a 10 ml saturated plate at 1.2K for 10 minutes and wash with 10 ml HBSS. Resuspend in 1 ml 90% Fetal Calf Serum 10% DMSO. Place in foam container overnight at -80°C. Then transfer to liquid nitrogen.

• To thaw, quickly warm the cryovial to 37°C until just thawed. Add 10 ml prewarmed medium dropwise, and spin 10 minutes at 1.2K to pellet. Resuspend in 10 ml medium and incubate at 37°.