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sect73
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).