Random primer labeling of polyA+RNA | Practical Molecular Biology

Random primer reaction.

prepare in nuclease-free tube on ice the following reaction mix:

d(N)₆ primer (50 OE ~1.7µg/µl) - 1µl
polyA⁺RNA ~1.5µg
H₂O that final volume in 5 will be 30ml;

to anneal primer, heat mixture to 70^oC 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 37^oC for 2 min;
add 1.5ml (300units) SuperScript II and mix;
incubate 1h at 37^oC;
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);

cDNA purification.

add 3.5µl 3N NaOH, mix;
incubate mixture at 68^oC 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!).

Hybridization.

pre-hybridization in HSB(f+) at 50^oC 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 45^oC for about 40h (2x ON) (but note, that we newer checked the intensities of signals for shorter hybridizations!);

Washing.

3 washings for 10 min in (1xSSC, 0.1%SDS) at normal temperature (it does not matter, if temperature will be 65^oC, so, we generally use normal temperature buffer in hot hybridizer);

2 washings for 20-30 min in (0.2xSSC, 0.1%SDS) at 65^oC (preheated buffer (!), it would be better first to rinse bottles quickly couple of times just to change buffer and preheat the bottle).

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 -20^oC.