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Transfection of Eukaryotic Cells
Posted on Wednesday, November 19, 2003
Description
Transfection protocol I have used recently
Procedure
1) Plate out near confluent cells @ 0.5 - 1 x 106 cells/90 mm plate. 2) Cells re-feed with 9ml medium.
3) 3hrs later, prepare the following 2 solutions at room temperature:- For each 90mm plate :-
TUBE A : 500ul 2 x HBS.
TUBE B : 62ul 2M CaCl2.
20ug carrier DNA (sheared salmon sperm DNA). 1-10 ug Plasmid DNA. sterile, nanopure water upto 500ul. 4) Add contents of tube "B" dropwise to tube "A". Add very slowly & gently to prevent large blobs of ppt. "B" must always be added to "A". N.B.: pH of the solutions at this stage is critical. The ppt. should be fine and not lumpy in appearance.
5) Incubate solution for 15 mins at room temperature.
6) Set up a control plate with no DNA - replace with water.
7) Remove plates individually from incubator. Aliquot 1ml of calcium phoshate/DNA ppt. onto each plate, mix gently and immediately return to the incubator.
8) After 16 - 24 hours, wash cells with serum free medium. Refeed cells with complete medium.
9) 48 Hours after transfection, change cells to DMEM + 10% FCS containing G418 (0.8 - 1.0 mg/ml).
10) Change medium on cells every 3 days - medium as above.
11) After 4-6 weeks of selection (colonies 3-5mm diameter), clones were trypsinised using cloning rings.
12) Aspirate off as much medium as possible.
13) Place cloning ring into vaseline (sterilized by autoclaving), so that the edges are covered.
14) Place ring around a suitable clone, fill with trypsin (0.25% in Ca/Mg-free PBS).
15) Incubate for 1-2 minutes allowing cells to detach.
16) Add cells to 1 well of a 24 multi-well plate containing 2ml of medium.
Recipes
10x HBS:- 1.4M NaCl. 250mM HEPES. 10mM NaHPO4. Adjust to pH 7.12 with 1M NaOH, and filter sterilise prior to use).
G418 (Stock 50mg/ml in PBS, can be stored at -20°C). 2M CaCl2
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