Random Hexamer Primed Probe

Protocol for making random primed probes for Northern Blots. It uses Klenow fragment to extend random DNA hexamers in the presence of an alpha labelled nucleotide. Plug in the concentration of your target DNA (blue box), and adjust the dNTP label volume if needed to account for decay.

Reagents Needed:
30-100 ng Target DNA (i.e. a restriction fragment or purified PCR product)
50 uCi alpha-labelled dNTP 3000 Ci/mmol
Random Hexamers - 1 to 2.5 mg/ml
10x Klenow Fragment Buffer (KFB)
3 dNTP mix 0.5 mM (all nucleotides except the one used for label)
BSA 0.5 mg/ml
DNA Pol I Klenow Fragment, 5 units
Assemble Pre Mix, denature, add components of Rxn Mix, incubate 2-4 hrs, stop rxn, purify.

Probe Name:
Pre Mix 12 ul Target Volume   ng DNA 30-100 ng
ul DNA ng/ul
2 ul pd(N)6 2.2 mg/ml (1-5 ug)
ul Water or TE  
ul Total
Boil 2-3 minutes
Rxn Mix 12.5 ul Target Volume
2.5 ul 10x KFB
2.5 ul dNTP mix 0.5 mM
2.5 ul BSA 0.5 mg/ml
Typical Label ul alpha-32P-dNTP ref. date
Vol. 5 ul ul Total 10x KFB
500 mM Tris HCL pH 7.5
Total 24.5 ul Pre + Rxn 100 mM MgCl2
add 1 ul Klenow 5 u/ul 10 mM DTT
Incubate at Room Temp. 2-4 hrs

Stop reaction with 1 ul 0.5 M EDTA
add  1 ul tRNA 10 mg/ml
    75 ul TE
remove 1.5 ul for counting/calculating specific activity
Purify by Gel Filtration (G50 in a Pasteur Pipet or a spin column).

Chris Seidel