OLIGOLABELLING DNA: RANDOM PRIMER METHOD

OLIGOLABELLING DNA: RANDOM PRIMER METHOD

to 9 ul of DNA (dilute with ddHO, approx. 100 ng DNA desired)
add 1.25 ul of hexamers (random primers)
boil 2', ice immediately
add 5 ul of HEPES
5 ul of nucleotides
1 ul of BSA (40 ng/ml)
.5 ul of Klenow
mix on ice
at room temp, add 5 ul of 32P-dCTP
incubate room temp x overnight
to separate unincorporated nucleotides:
dilute labelling reaction with 180 ul TE
run through 1 ml spin-column packed fresh with biogel
boil probe before adding to hybridizations
NOTES:
  1. see preparation of nucleotide solution for oligolabelling for content of nucleotide solution.
  2. spin-columns are made with bio-gel pre-swollen with TE
    wash columns once with 200 ul TE prior to use
    pack columns in clinical centrifuge at approx. 1/4 speed.

PREPARATION OF NUCLEOTIDE SOLUTION FOR USE IN OLIGOLABELLING

NEED STOCK SOLUTIONS OF:
dATP
dTTP each dNTP stock should be: 100 mM dNTP
dGTP 1 mM EDTA
pH= 7.0
(adjust pH with Tris base)
NUCLEOTIDE STOCK SOLUTION IS:
2 mM of each dNTP
25 mM MgCl2
250 mM Tris-Cl (8.0)
50 mM beta-mercapto-ethanol
TO MAKE 1 ml OF NUCLEOTIDE STOCK SOLUTION:
20 ul of each .1M dNTP
25 ul 1 M MgCl2
250 ul 1 M Tris-Cl (8.0)
3.5 ul beta-mercapto-ethanol
661.5 ul ddH2O
NOTE:
  1. freeze dNTP stocks in 20 ul aliquots for future use.