Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Digoxigenin labelled probe synthesis
Tautz and Pfeifle as modified by members of the Hoffmann lab

Use Buffer 5 (10x hexanucleotide mix) and Buffer 6 (10x nucleotide mix) from BMB Genius kit OR


10x hexanucleotide mix              10x nucleotide mix
0.5 M Tris 7.2 1 mM dATP 0.1 M MgCl2 1 mM dCTP 1 mM DTT 1 mM dGTP 2 mg/ml BSA 0.65 mM dTTP 3 mg/ml pDN6 (Pharmacia) 0.35 mM digoxygenin- 11-dUTP
Also needed:
pDN6 random primer (Pharmacia) at 25 mgs/ml (Genius kit pDN6 is not concentrated enough; 1 O.D. unit = 20 ug/ml)
DNA fragment chopped into pieces of < 400 base pairs
Klenow fragment of DNA polymerase I
tRNA at 25 mgs/ml
EDTA 0.5 M pH 8
PBT (PBS + 0.1% Tween 20)(PBS = 10 mM KPO4; 140 mM NaCl pH 7.2)
Centricon 10 (Amicon)

1) Combine: 100 ng (embryo probes) or 400 ng (disc probes) fragmented DNA
2 ul pDN6 (at 25 mgs/ml)
deionized H2O to 9 ul

2) Boil for 2 min.; quick chill in ice water

3) Add : 2 ul 10x hexanucleotide mix
2 ul 10x nucleotides
6 ul H2O
1 ul Klenow

4) Place in 16oC water bath overnight.

5) Stop by adding: 4 ul of EDTA and placing at 80oC for 15 min.

6) Remove excess nucleotides by adding 2 ul of tRNA and 2 mls of PBT and put in Centricon 10 for 50 min. at 6000 rpm.

7) Add another 2 mls of PBT and spin in Centricon again.

8) Centrifuge probe out of Centricon and add PBT to 100 ul. Add 100 ul of deionized formamide. The probe concentration is now 0.5 ng/ul (embryo probes) or 2 ng/ul (disc probes). Store dig probes at -20oC. Use 17 ul of probe (denatured for 5-10 min at 85oC) per100 ul hybridization for both embryos and discs.

 

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