氯化钙法转染 with BES transfections

BES transfections

1.The day before transfection pass the cells 1:5 or 1:8 (depending on the cell type).

Next day the cells should be ideally 30-40% confluent, which is optimal for

transfection. Cells can be transfected the same day they’re plated (at least 3 hrs

hrs after), but the efficiency of transfection may be lower.

2.Replace the medium (10 ml per p100, DMEM + 10%FCS + 1% pen/strep) at least

2 hrs before transfection (this is not absolutely crucial, but will enhance the

efficiency of DNA uptake).

3.Mix the following components in a Falcon 2054 tube (the order of addition is

important):

a.10-12 µg (12µg is better) of purified DNA (maxi-prep quality, ideally

CsCl gradient purified)

b.Add 450µl of deionized sterile water

c.Add 500µl of 2xBES pH 6.95. Swirl to mix. The absolute pH optimum

often has to be determined empirically, e.g. prepare buffers with pH

ranging from 6.85-6.98.

d.Add dropwise 50µl of 2.5M CaCl2 and shake vigorously

e.Allow the precipitate to form for 10 min. Shake up precipitate every few

minutes.

4.Add precipitate to cells dropwise and uniformly all over the plate. Swirl plate.

5.Incubate cells with precipitate at 3% CO235 ˚C overnight (not critical, you can

use 5% CO2, 37 ˚C). The precipitate should be very fine, not chunky.

6.Following day: Wash cells twice with PBS and feed with fresh medium.

7.Harvest cells after 48hrs (or 72 if it’s more convenient).

-20˚C.