DNA TRANSFECTION OF EUKARYOTIC CELLS USING CALCIUM PHOSPHATE

 

Stock Solutions

1. 2M Cacl2 Merck. Filter (to remove particulates), autoclave and store at -20¡ in aliquots.

2. 2 x HEBS gm/500ml

NaCl 8.0

KCl 0.38

Na2HPO4.7H2O 0.19

glucose 1.0

Hepes 5.0

Adjust pH to 7.05 ± .05 with NaOH

Autoclave

Store frozen in aliquots

3. 1 X HEBS/15% glycerol. 50ml 2 x HEBS )

15ml glycerol )

35ml DDW )

Autoclave and store at -20¡ in aliquots

4. Carrier DNA. Mouse liver DNA, sheared at 90ug/ml in 0.2XSSC by passing x2 through a 20 gauge needle; filtered through a 4.45um filter.

Transfection protocol

1. Plate cells at 1 x 106 in 10cm dishes

2. Next day perform transfection.

Preparation of CaPo4 Precipitates

1. Set up two rows of tubes - A & B

In tube A place:

15ug of plasmid DNA (linearized, phenol extracted, precipitated and resuspendedat 1ug/ul in sterile

0.2X SSC

69ul 2M CaCl2

460 0.2X SSC

In tube B place 550ul 2X HEBS

2. Using two autopipettes and 1ml pipettes have tube B bubbling whilst slowly adding contents of tube A.

3. Stand 15-20 mins., while precipitate forms, giving a milky fine ppte.

4. Carefully add precipitate dropwwise to 10cm dish of cells whilst maintaining the pH of cultures.

5. Leave 3-4 hrs in CO2 incubator (Can be left o/n).

6. Glycerol Shock:

Aspirate medium

Wash by adding ~3ml medium then aspirate

Add 2.5ml 15% glycerol in HEBS.

Sit 4 mins at room temp. then aspirate

Wash with 5ml medium per plate

Add fresh medium

NOTE: Cell are fragile and only loosely attached at this stage. Hand very gently. If ppte was left on cells o/n do the splits next day.

Next day:

7. Trypsinise to harvest cells. Recover cells in medium containing antibiotic and divide directly in the ratio 3/5th's and 1/20th into two 10 cm plates, respectively.

8. Feed every 2-3 days with medium containing antibiotic (once a week when most cells have been killed).