反応 #632836

反応方程式

dimethylsulfoxide

DMSO

DMSO

HeBES

CaCl2

DMSO

PBS

→

Calcium Phosphate

溶媒

water

反応条件

後処理

1. 1
   その他The HeBES was bubbled with a pipettor
2. 2
   workup.ADDITIONwhile adding the DNA-calcium mixture with a Pasteur pipette
3. 3
   workup.ADDITIONThe mixture was mixed by vortex for 5 seconds
4. 4
   その他prepared for transfection in F12 medium
5. 5
   workup.WAITthen incubated at 37° C. for four hours
6. 6
   洗浄washed two more times in PBS
7. 7
   workup.ADDITIONTen (10) ml of αMEM/5% FBS/HT was added
8. 8
   workup.WAITthe plates were incubated at 37° C. overnight

実験手順

Seventy-five micrograms (75 μg) of DNA were dissolved in 1.35 ml water in a 50 ml conical tube. One hundred and fifty microliters (150 μl) of 2.5 M CaCl2 were added, and this DNA-calcium mixture was added dropwise to 1.5 ml of 2× HeBES (HEPES buffered saline) in a 50 ml conical tube. The HeBES was bubbled with a pipettor while adding the DNA-calcium mixture with a Pasteur pipette. The mixture was mixed by vortex for 5 seconds and incubated at room temperature for 20 minutes. One milliliter (1 ml) of the DNA-calcium mixture was distributed evenly over each culture dish of adherent cells, grown and prepared for transfection in F12 medium as described above, and the cultures then incubated at 37° C. for four hours. After incubation, the plates were aspirated, and 2 ml of 10% dimethylsulfoxide (DMSO) in F12 medium was added to each plate (DMSO shock treatment). The DMSO shock continued for one minute after which the DMSO was diluted by the addition of 5 ml of PBS (phosphate buffered saline) to each plate. The plates were aspirated and washed two more times in PBS. Ten (10) ml of αMEM/5% FBS/HT was added, and the plates were incubated at 37° C. overnight.

出典