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Random primer labeling of polyA+ RNA

 

    Random primer reaction

  1. prepare in nuclease-free tube on ice the following reaction mix:
  2. d(N)6 primer (50 OE ~1.7µg/µl) - 1µl
    polyA+RNA ~1.5µg
    H2O that final volume in 5 will be 30ml;

  3. to anneal primer, heat mixture to 70oC for 5 min and chill on ice for 2 min;
  4. if necessary, collect the contents of the tube by brief centrifugation and add:
  5. 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.

  6. mix contents of the tube and incubate at 37oC for 2 min;
  7. add 1.5ml (300units) SuperScript II and mix;
  8. incubate 1h at 37oC;
  9. chill on ice;
  10. 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

  11. add 3.5µl 3N NaOH, mix;
  12. incubate mixture at 68oC for 20 min;
  13. add 10µl 1M TrisCl (pH 7.4) and mix, then add 10.5µl 1N HCl and mix;
  14. change buffer and remove unincorporated dNTP's by centrifugation through G-50 (or analogous gel-filtration column);
  15. 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!).
  16. Hybridization

  17. pre-hybridization in HSB(f+) at 50oC for at least 1 hour;
  18. denature the probe in boiling water bath for 1 min (note: it is unnecessary if you just purify probe on G-50);
  19. substitute pre-hybridization buffer for hybridization one (the same buffer, but with the probe);
  20. hybridization at 45oC for about 40h (2x ON) (but note, that we newer checked the intensities of signals for shorter hybridizations!);
  21. Washing

  22. 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);
  23. Conc.Stock1L
    SSC1x20x50ml
    SDS0.1%10%10ml
    H2O mQ940ml
  24. 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).
  25. Conc.Stock1L
    SSC0.2x20x10ml
    SDS0.1%10%10ml
    H2O mQ980ml

    Stripping of membranes

  26. preheat the 0.5%SDS solution to boiling (be especially careful with the filtered SDS!!! It likes to start boiling with explosion);
  27. dip membrane in the boiling SDS and boil further for 1 min;
  28. take out vial with membranes from heater and leave it on the rocker for 3-5 hours;
  29. wrap wet(!) membranes on SaranWrap, make control exposition and keep membranes on -20oC.


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    Solutions

    C - mix

    20x, (store at -20oC)

    Conc.Stock100µl
    dATP20mM100mM20µl
    dGTP20mM100mM20µl
    dTTP20mM100mM20µl
    dCTP0.12mM5mM2.4µl
    H2O mQ37.6µl

    HSB(f+)

    high SDS hybridization buffer(store the main stock at 4oC, working - NT)

    Conc.Stock500ml
    SDS7%solid35g
    Formamid50%100%250ml
    283.3g

    SSC5x20x125ml
    145.3g

    Bloc. Reagent2%solid10g
    NaP (pH 7.0)50mM1M25ml
    27.6g

    N-Lauroylsarcosine Na0.1%10%5ml
    5.1g

    Salm.Sperm DNA50µg/ml10µg/µl2.5ml
    H2O mQ49ml

    p=1.111

  • Blocking Reagent - is the powder from "Roche Diagnostics GmBH; former Boehringer Mannheim" #1096176. Instead of 2% Blocking Reagent one may use 10x Denhardt or 2% non-fat dry milk.
  • it is possible to dissolve the Blocking Reagent only in high strength buffer (in this particular case in H2O+SSC+NaP).
  • Salmon sperm DNA should be shredded and denaturated.
  • We use the following reagents:

    d(N)6 primerPharmacia#27-2166-01
    SuperScript IIGibcoBRL#18064-014
    FSB, DTTGibcoBRL5x First Strand Buffer,
    supplied with SuperScript II
    RNase BlockGibcoBRL#10777-019
    dNTP'sPharmacia#27-2035
    [a33P]dCTPAmersham#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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Last modification: 30/07/13

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