NICK TRANSLATION OF dsDNA WITH BIOTINYLATED OR DIGOXIGENIN dUTP
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<center> <font>NICK TRANSLATION OF dsDNA WITH BIOTINYLATED OR DIGOXIGENIN dUTP</font></center>
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
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x µl bidestilized water till endvolume is 50 ml
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5 µl 10xnick translationbuffer
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5 µl nucleotide mix (endconcentration = 2.5 mM):
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for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM):
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3 µl stock solution + 27 µl bidest (1)
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25 µl of each (1) +25 µl H20 = 100 µl mix
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for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM):
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2.5 µl bio-16-dUTP (0.4 mM, ready for use)
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5 µl 100 mM DTT
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x µl DNA (usual 1 mg disolved in 1 ml TE)
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5 µl DNase I (stock 1 mg/ml, dilute 1/1000 in H20, prepare fresh every time)
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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 :