For the use with FISH (fluorescent in situ hybridization) we always label entire plasmid DNA and do not cut out the insert.
1. mix together
- x µl bidestilized water till endvolume is 50 ml
- 5 µl 10xnick translationbuffer
- 5 µl nucleotide mix (endconcentration = 2.5 mM):
- for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM):
- 3 µl stock solution + 27 µl bidest (1)
- 25 µl of each (1) +25 µl H20 = 100 µl mix
- for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM):
- 2.5 µl bio-16-dUTP (0.4 mM, ready for use)
- 5 µl 100 mM DTT
- x µl DNA (usual 1 mg disolved in 1 ml TE)
- 5 µl DNase I (stock 1 mg/ml, dilute 1/1000 in H20, prepare fresh every time)
- x µl DNA polymerase (endconcentration must be = 30 U)
3. stop the reaction with 5 µl 0.5 mM EDTA (pH 7.4)
4. add to each tube:
6. this mixture overnight or 1 hr at -70°C (precipitation)
7. centrifuge 30 min at 14000 rpm at 4°C
8. remove supernatans (vacuumpomp) and dissolve in appropiate volume :
