Biology Reference
In-Depth Information
Box 17.1
Ca
3
(PO
4
)
2
transfection
- 2.5
10
5
cells are seeded in a 10 cm dish in 10 ml DMEM containing 10% FCS and
supplemented with
L
-glutamine (3 mM) and penicillin (100 units/ml)/streptomycin
(100 mg/ml).
- The following day, medium on the cells is refreshed 1 h before transfection with 9 ml DMEM
supplemented with 10% FCS.
-10mg of DNA is used for transfection of cells in one 10 cm dish. If more than one plasmid is
used for transfection of the same plate, for example, when studying protein-protein
interactions, different plasmids with cDNA encoding the different proteins are need. Take
the same amount of encoding plasmids for the different setups, and equalize DNA amounts
among different plates by adding parental plasmid (¼“mock” plasmid), so that the total
amount of plasmids used for each plate is the same (¼10 mg).
- Prepare per dish the following mix (in the mentioned order):
100 ml 2 M CaCl
2
10 ml DNA (1 mg/ml)
390 ml 0.1 TE (1 mM Tris/HCl pH 7.6; 0.1 mM EDTA pH 8)
- Add the mix dropwise to 500 ml2 BS/HEPES (25 mMHEPES; 274 mMNaCl; 10 mMKCl;
1.5 mM Na
2
HPO
4
; 12 mM dextrose pH 7.05) and aerate the complete mixture. Adding the
calcium/DNA mixture to the phosphate buffer will result in small precipitates.
- Add the mixture to the cells and mix gently.
- Refresh the medium after at least 4 (up to 24) hours with DMEM 10% FCS.
PEI transfection
10
5
cells are seeded in a 10 cm dish in 10 ml DMEM containing 10% FCS and
supplemented with
L
-glutamine and penicillin/streptomycin.
- The following day, cells are refreshed with 9 ml DMEM supplemented with 2% FCS.
- Prepare PEI stock solution:
Dissolve PEI (high molecular weight, water-free (25 kDa branched), Sigma Aldrich:
40,872-7): 100 mg/ml in Milli-Q water and homogenize.
Dilute further to 1 mg/ml and adjust pH to 7 with HCl.
Filter sterilize and aliquot (can be stored at
2.5
20
C for
>
year).
Alternatively several companies sell this product.
- Prepare per dish the following mixes:
Mix 1: 10 ml DNA (1 mg/ml)þ490 ml serum-free medium (without antibiotics) (¼SFM).
Mix 2: 25 ml PEIþ475 ml SFM.
- Add mix 2 to mix 1, vortex, and incubate for 10 min at room temperature.
- Add the mixture to the cells and mix gently.
- Refresh the medium after 6 h with DMEM 10% FCS.
Lipofection
- 2.510
5
cells are seeded on a 10 cm dish in 10 ml DMEM containing 10% FCS and
supplemented with
L
-glutamine and penicillin/streptomycin.
- The following day, cells are refreshed with 9 ml SFM.
- Prepare per dish the following mixes:
Mix 1: 10 ml DNA (1 mg/ml)þ490 ml SFM
Mix 2: 20 ml Lipofectamine 2000 (Invitrogen)þ480 ml SFM
- Add mix 2 to mix 1, vortex, and incubate for 45 min at room temperature.
- Add the mixture to the cells and mix gently.
- Refresh the medium after 6 h with DMEM 10% FCS.
