Calcium Phosphate Transfection of 293T                         

                                   

 

I. Reagents/solutions

 

1. transfection media: 

 

293T cells about 50% confluent. If the cells are too confluent they will not transfect well. You do the transfection in the normal cell medium.

 

2. 2X HeBs    

 

The quality and accurate pH of this solution is of the utmost importance for efficient transfection. We use the highest quality chemicals, and keep them separate from our normal dry chemical stocks. These are labeled tissue culture only and never are any lab implements allowed into the containers.

 

                                    final conc         for 200 ml       supplier

 

NaCl                            274 mM          3.2 g                Baker # 3624-05; Mallinckrodt

KCl                             10 mM            142 mg            Mallinckrodt # 6858

Na2HPO4.7H2O        1.4 mM           76 mg              Mallinckrodt # 7914; 268g/mol

dextrose (D-glucose)   15 mM            540 mg            Baker # 1916-01; 180g/mol

Hepes (free acid)         42 mM            2 g                   Calbiochem Ultrol # 391338; 238g/mol

 

 

- add components to 180 ml. water (we use only ddH2O straight from the purifier, and only glassware that is tissue culture only, never having seen soap)

- pH with 1 or10N NaOH (we make special NaOH only used for this)

- pH very carefully to 7.02 – use most accurate pH meter you have.

- bring to 200mL with ddH20

- remove 25 mL and save

- pH remainder to 7.04 (with 1N NaOH); remove 50mL and save

- pH to 7.06; remove 50mL and save

- pH to 7.08; remove 50mL and save

- pH to 7.10; this is the last 25 mL

- filter sterilize each pH aliquot separately in cell culture hood

- aliquot to 1ml aliquots (eppendorf tubes)

- store at -20C; thaw individual aliquots as used. I usually do not reuse unless for nonneuronal cells.

 

- note, as for any CaP transfection, greatest efficiency is obtained with a fine, ÒsandyÓ ppt.  This is critically dependent on the pH of the HeBS, so it is best to:

            - use an accurate pH meter

            - standardize the pH meter (with pH 4.0 and 7.0 standards) repeatedly, until                                     standards are read precisely

- as indicated above, make and test multiple batches of 2X HeBS, with slightly different pHÕs (eg., pH 7.02-7.10); test each, use best, toss the rest, or use for nonneuronal cells

- seems to be good to make fresh 2X HeBS at least every few months

 

 

3. 10X CaCl2

 

            Make 2.5M in ddH20. Filter sterilize and store.

            - this does not seem to be one of the finicky steps.

 

 

II. Protocol

 

1. Prepare cells

 

Should be about 50% confluent on day of transfection. Leave on the culture medium. Can also have cells in suspension after just having split them.

 

2. make calcium phosphate/DNA precipitate:

 

a. variables

 

- volume of ppt

            6 well plate: 120uL/well

            100mM plate: 1.2mL/plate

 

- amount of DNA :

            6 well: 2-5ug (eg., for RNAase protection)

            100mM plate: 5-20ug (RNAse protection/expression)

 

b. recipe: eg., for 120ul precipitate/well (scale up or down accordingly):

           

1) in a 15 ml polystyrene pop-cap tube (Falcon 2058), mix DNA, ddH2O, and CaCl2 .

 

            9-18ug CaRF DNA (2-4ug/well x 4.5 wells)

            9ug GFP (2 ug/well x 4.5 wells)        

54uL 10X CaCl2 (2.5M)  (1/10 final volume)

            ddH2O (tissue culture sterile, direct from purifier) to 270uL (60uL/well x 4.5)

 

2) aliquot an equal volume 2X Hepes-Buffered Saline (HeBS) to second tube for each ppt (270uL, in this case)

 

3) thoroughly mix DNA/CaPO4 mixture and add to 2X HeBS dropwise with pipetman.

           

There are many schools of thought on exactly how you should make this mixture. Some people bubble the DNA through the HeBS, some swirl the tube. I am in the camp that holds the pipetter with DNA mix above the HeBS and then just slowly drops one drop at a time on top of the HeBS. Different mixing styles give different size precipitates that will influence the amount of ppt and time of transfection you should use. Therefore, each person must optimize these parameters his/herself since everyone mixes slightly differently.

 

4) let ppt form 5Õ RT

            For neurons, we always let the ppt form for longish periods of time before putting on the cells. For nonneuronal cells, however, mixing the ppt then applying directly to cells seems to work best. If you are in the mood to optimize you could test for your cells the effects of changing the time of ppt formation.

 

            As to forming the ppt in the dark, we know of no good reason why that is supposed to matter, though all books say that it does. We frequently form ppts in the light and have not noticed a difference.

 

3. Add ppt to plates

 

- drip evenly over surface, with pipetman

- careful as you return pipetter to tube for each aliquot. Try to minimally disturb ppt.

- return plate to 10% CO2 incubator. Leave O/N. Cells are ready to harvest the next am.

 

If you are going to make nuclear extracts, change the medium after O/N in ppt. and leave 24 hours to dissolve CaPO4 ppt.