Good thing takes time

using PGEM3/4, the size is about 4kbp. Therefore, using 10ug is about 3.8E-12 mole.

but using AvaI to cleavage, the end is GGCC, therefore is equal to 4 times of DNA molarity, is about 3.8X4= 16E-12 mole.

for 6000Ci/mmole dCTP, is about 3.3 E-12 mole/uL.

1. AvaI cleavage:

pGEM3/4z 10ug(50uL)
NEB4 20uL
AvaI 5uL
H2O 125uL % 37 degree/4hours %
% check by agarose for completion %
% 95 degree/5min;to remove the enzyme from the end %

2. gamma-32p dCTP(6000Ci/mmol) labeling

0.1 mM dGTP
10 u Klenow % room temp/ 40min %

3. ammonium acetate precipitation (removing free nucelotide)

1/2V 7.5M (NH4)2oAC
2.5-3V 100% EtOH % -70 degree/1hour %
% 14,000rpm./4 degree/ 25min/ %
% 70% EtOH wash 2X %
% Dissolve in 200 uL EB buffer %