2 washings for 20-30 min in (0.2xSSC, 0.1%SDS) at 65oC (preheated buffer (!), it would be better first to rinse bottles quickly couple of times just to change buffer and preheat the bottle).
Random primer reaction.
- prepare in nuclease-free tube on ice the following reaction mix:
d(N)6 primer (50 OE ~1.7µg/µl) - 1µl
H2O that final volume in 5 will be 30ml;
- to anneal primer, heat mixture to 70oC for 5 min and chill on ice for 2 min;
- if necessary, collect the contents of the tube by brief centrifugation and add:
5x FSB (first strand buffer) - 6µl
0.1M DTT - 3µl
C-mix, 20x - 1.5µl
RNase Block - 1µl
a[33P]dCTP (10µCi/µl) - 7*µl
* it is just a sample, you can use another quantities of labeled nucleotide.
- mix contents of the tube and incubate at 37oC for 2 min;
- add 1.5ml (300units) SuperScript II and mix;
- incubate 1h at 37oC;
- chill on ice;
at this point it is possible to determine the incorporation rate by TCA precipitation (normally, it is practically quantitative) or to go directly to the purification of cDNA (note: if in TCA precipitation experiment you use hand-held monitor, GFC filters should by dry, because water dramatically suppress the 33P signals);
- add 3.5µl 3N NaOH, mix;
- incubate mixture at 68oC for 20 min;
- add 10µl 1M TrisCl (pH 7.4) and mix, then add 10.5µl 1N HCl and mix;
- change buffer and remove unincorporated dNTP's by centrifugation through G-50 (or analogous gel-filtration column);
- estimate the incorporation in reaction by direct counting (compare the "counts" from both column and vial with eluted material and from eluted material alone; estimation is only qualitative!).
- pre-hybridization in HSB(f+) at 50oC for at least 1 hour;
- denature the probe in boiling water bath for 1 min (note: it is unnecessary if you just purify probe on G-50);
- substitute pre-hybridization buffer for hybridization one (the same buffer, but with the probe);
- hybridization at 45oC for about 40h (2x ON) (but note, that we newer checked the intensities of signals for shorter hybridizations!);
- 3 washings for 10 min in (1xSSC, 0.1%SDS) at normal temperature (it does not matter, if temperature will be 65oC, so, we generally use normal temperature buffer in hot hybridizer);
Stripping of membranes.
preheat the 0.5%SDS solution to boiling (be especially careful with the filtered SDS!!! It likes to start boiling with explosion);
dip membrane in the boiling SDS and boil further for 1 min;
take out vial with membranes from heater and leave it on the rocker for 3-5 hours;
wrap wet(!) membranes on SaranWrap, make control exposition and keep membranes on -20oC.