This is a cached page for the URL (http://grimwade.biochem.unimelb.edu.au/bowtell/cellbiol/sect73.htm#DNA TRANSFECTION OF EUKARYOTIC CELLS). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
sect73

[Previous] [Top] [Next]

sect73

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).

[Previous] [Top] [Next]


This page is maintained by David Bowtell (bowtell@ariel.ucs.unimelb.edu.au) using HTML Author. Last modified on 10/24/95.