C- Method for Making Probes
Kim Nakamura, 1999
1.
Combine 20-40 ng of isolated fragment with H2O to a final volume of 7.5 µl.
Boil 5 min then put on ice for 5 min.
2.
Immediately add:
10 µl 2.5 X C- buffer
1 µl 10 mg/ml BSA
6 µl [32P]  dCTP
1 µl Klenow
Mix
Leave at RT for 2+ hrs.
3.
Add 75 µl TE and load onto spin column containing G50 in TE (See below for
spin columns).
Spin at setting 4 for 2 min in silver mushroom centrifuge.
Measure volume and dot 1 µl on two pieces of filter paper.
Count one piece in scintillation counter.
Wash other piece with:
NaPO4 buffer, 3 X 5 min
H2O, 3 X 5 min
EtOH, 3 X 5 min.
Let dry and count in scintillation counter. Compare counts. This determines the
efficiency of the probe.
4.
Boil probe for 5 minutes, then put on ice for 5 min.
Add ~5 ml of Hybridization buffer with 50% formamide
Incubate O/N at 42 C.
Pour off probe into falcon tube and freeze at -80 C. Can reuse probe within 2
weeks. After that the 32P has degraded to half.
Start washes on blot.
2.5 X C- Buffer (5 mL vol):
2.5 ml 1 M Hepes pH 6.6
25 µl 1 M MgCl
10 µl -mercaptoethanol
625 µl 1M Tris pH 7.5
30 µl 10 mM dATP
30 µl 10 mM dGTP
30 µl 10 mM dTTP
1750 µl sddH2O
Spin columns:
Stuff a small bit of siliconized glass wool into a 1 cc syringe. Smash it to the bottom with
the plunger. Set syringe into a 15 ml falcon tube. Load a slurry of G50 in TE into
syringe. Fill up to the 1 cc mark. Wash with 5 volumes of TE. Transfer syringe to
polypropylene tube with snap cap. Load probe onto column, put on snap cap and spin. 