* 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);
cDNA purification
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!).
Hybridization
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!);
Washing
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);
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).
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.
5x First Strand Buffer, supplied with SuperScript II
RNase Block
GibcoBRL
#10777-019
dNTP's
Pharmacia
#27-2035
[a33P]dCTP
Amersham
#AH9905
Comments
We use 30µl reaction volume for labeling reaction with ~1.5µg of mRNA. It seems, it is still possible to use 2-5 times less quantities of mRNA.
It is possible to use oligo(dT) primer. In this case one should take ~0.7µg (dT)6 per 1.5µg mRNA.
Not only mRNA may be used as a template. One can take first strand cDNA (or double stranded cDNA) and perform the normal Random Primer labeling reaction. In this case it is possible to use smaller quantities of template, because labeled nucleotide should not be diluted.
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