Binding reaction:
Mix in a 50µl final volume:
0-25µl protein extract in HEMG (see below)
10µl 10% polyvinyl alcohol (store 4°C)
1µl 1M Hepes pH 7.6 (pHed with KOH)
~2fmoles of end-labelled DNA probe (~20cps on minimonitor)
competitor DNA (see below)
ddH2O to a final volume of 50µl
If the amount of protein used is less than 25µl, add 0.1M HEMG to make up the
difference. Mix components gently and incubate ~10 minutes. Incubation on ice is
usually required when dealing with relatively crude protein extracts which contain
endogenous nucleases and/or phosphatases. With purified proteins the incubation
temperature can be increased.
It is advisable to add non-specific competitor DNA, such as sonicated calf
thymus DNA, synthetic poly d(I-C) or poly d(A-T) (Pharmacia) to the binding reaction.
The optimal competitor concentration needs to be determined empirically, however
0.1-0.5µg of sonicated calf thymus DNA or 0.01 OD units of synthetic DNA are usually
adequate.
DNAaseI digestion:
To the binding reaction add 50µl Ca/Mg solution (10mM MgCl2, 5mM CaCl2) at
room temperature and 1-10µl of DNAaseI solution (see below). Mix quickly and
incubate at room temperature for 1 minute. Stop the reaction by adding 100µl of stop
solution (0.2M NaCl, 20mM EDTA pH 8, 1% SDS, 0.25 mg/ml carrier RNA; store at
room temperature) and vortexing immediately. Digestion of three samples can be carried
out simultaneously with a bit of practice.
Samples are then extracted with 200µl of 1:1 phenol:chlorophorm and
precipitated with 1ml of 100% ethanol. Pellet is washed with 70% ethanol, dried and
resuspended in 6µl of formamide dye. Run on 6-8% sequencing gel.
HEMG: 0.1M KCl
25mM Hepes pH 7.6 (pHed with KOH)
0.1 mM EDTA pH 8
12.5mM MgCl2
10% glycerol
1mM DTT (fresh)
DNAaseI: Make a 2.5mg/ml DNAaseI (Worthington or Cooper Biomedical, bovine
pancreas, DPFF, 2000U/mg) solution in ddH2O and freeze 2-5µl aliquots; keep at
-70°C. Make a fresh dilution of this stock solution in ice cold ddH2O and keep on ice.
For the no-protein control use ~1µl of a 1/1000 dilution. For crude protein extracts the
amount and dilution of DNAaseI needs to be determined; 5µl of a 1/100 dilution are
usually adequate.