Radioactive Oligo labeling of DNA Keep minimize time working with 32P dCTP and use shield to protect.
The procedure is based on the Klenow fragment, or large subunit, of DNA polymerase I. Klewnow retains the 5' - 3' polymerase activity but lacks the 5' - 3' exonuclease activity. Since Klewnow can not begin synthesis without a primer, a synthetic set of primers (6 bp random oligomers) are added to the single stranded DNA template. Some of these primers will have homology to most regions of DNA and can thus provide a point of initiation for synthesis.
- Mix probe stock briefly remove the desired amount of whole plasmid or insert stock (Good DNA amount in labeling DNA is 100 ng) and bring up to 31 µl with H2O in a MFT. If the stock is in low-melting agarous, boil the entire stock to melt for 1 min, then voltex prior to transfer and mix with H2O. If the DNA quantity in the stock is unknown, use 15 µl of the stock for labelling.
- Boil the MFT containing the probes for 10 min. in water bath to denature the DNA. Place on ice slurry containing NaCl for 30 sec., then place in a rack at room temperature.
- Immediately, add 19 µl Oligo reaction mixture to the MFT, and vortex lightly. Reaction mixture labeled "rxn mix", and placed on ice before boiling DNA at step 2.
Oligo reaction buffer :
Mixture of stock solution A, B, C in the ration of 1:2.5:1.5. Stored at -20 C.
Stock Volume 19 µl (for 1 MFT)
Oligo buffer 10.0 µl
BSA(10ng/µl) 2.0 µl
Klenow 2.0 µl
32P dCTP (3000 Ci/mmole)3 5.0 µl
- Incubate at 37 C for 1.5 hr (or longer). At room temperature for 2 hr or longer (or overnight, if desired).
- Stop reaction by adding 85 µl reaction stop buffer (RSB) to MFT.
- Remove the uncorporated 32P with sephadex G-50 spin columns (which see). After adding the reaction mix to the column, wash the tube with 100 µl of STE to the column.
- Assay the reaction by using a hand-held Geiger counter. A good reaction register between 1 and 2 kcps (thousand counts per sec) at a distance of about an inch from the phototube. If the reaction do not work, reading would be low, possibly 10-fold decease, indicating the probe would not be useful.
- Boil for 10 min, cool on ice for 1 to 2 min. Using the same tube from the spin column, dilute the entire probe with hybridization buffer immediately (should not be sit longer than 20 min).Solution A
Stock 2-mercaptoethanol (bME) 18 µl
DXTPs (A, T, G) 5 µl each
Solution O 850 µl
Store at -20 C
Solution O Conc. Stock 250 ml 100 ml
1.47 M Tris-Base 44.52 g 17.81 g
0.147 M MgCl2 7.47 g 2.99 g
Adjust pH to 8.0 with conc. HCl.
Solution B Conc. Stock 250 ml 100 ml
2 M Hepes 119.15 g 47.66 g
Adjust pH to 6.6 with 4 N NaOH. Store at 4 C.
Solution C Hexamer or oligo nucleotides. Add 55 µl H2O directly to the Pharmacia bottle which contains 50 units of lyophilized hexamer. Store at -20 C.
Klenow Dilute to 1 unit Klewnow fragment by adding klewnow buffer. Stock Klenow from BRL comes as 500 units in 84 µl. Dilute to 1 unit by adding 420 µl of klenow buffer to the 500 unit/84 µl stock. Store - 20 C.
Klenow buffer (250 ml)
7 mM Tris-HCl 0.211 g
7 mM MgCl2 0.355 g
50 mM NaCl 0.730 g
50 % Glycerol 125 ml
Add the above to about 80 ml H2O. Stir to dissolve. Adjust volume to 250 ml with H2O. Store at -20 C.
BSA Stock BSA from BRL comes as 50 mg/1000 µl. Add 4 ml of H2O for a final concentration of 10 mg/ml. Store at -20 C.
RSB (Reaction Stop Buffer) Conc. Stock Volume (ml)
10 mM 1 M Tris pH 8.0 1.0
2 mM 0.5 M EDTA 0.4
0.2 % 20 % SDA 1.0
STE (Sodium chloride and TE) Conc. Stock Volume (ml)
10 mM 1M Tris pH 8.0 10.0
1 mM 0.5 M EDTA 2.0
100 mM 5 M NaCl 20.0
Autoclave, and store at room temperature.