Archived Protocol

<html> <title>Nick translation of dsDNA with biotinylated or digoxigenin dUTP </title> <CENTER>NICK TRANSLATION OF dsDNA WITH BIOTINYLATED OR DIGOXIGENIN dUTP</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 <ul> <li>x µl bidestilized water till endvolume is 50 ml <li>5 µl 10xnick translationbuffer <li>5 µl nucleotide mix (endconcentration = 2.5 mM): <ul> <li>for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM): <li>3 µl stock solution + 27 µl bidest (1) <li>25 µl of each (1) +25 µl H20 = 100 µl mix </ul> <li>2.5 µl bio-16-dUTP (0.4 mM, ready for use) <li>5 µl 100 mM DTT <li>x µl DNA (usual 1 mg disolved in 1 ml TE) <li>5 µl DNase I (stock 1 mg/ml, dilute 1/1000 in H20, prepare fresh every time) <li>x µl DNA polymerase (endconcentration must be = 30 U) </ul> 2. mix carefully with finger and put for 2 hrs at 15°C (cryostat) <dd>for YAC and genomic DNA, check fragment length on 1.2 % agarose gel. <dd>The fragment length should be 300-400 bp for YAC <dd>and 600-1500 bp for genomic DNA (used in a CGH-experiment). <dd>If the fragment length is larger add more DNase I. 3. stop the reaction with 5 µl 0.5 mM EDTA (pH 7.4) 4. add to each tube: <dd>2.5 µl salmon sperm DNA (stock 10 mg/ml) <dd>2.5 µl yeast RNA <dd>5.5 µl 3 M NA-acetate (pH 5.6) <dd>137.5 µl icecold 100 % EtOH 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 : <dd>-repetitive probes in 100 µl 60 % form/SSCP <dd>-unique sequences in 50 µl 50 % form/SSCP <dd>-cosmids, YACS in 50 µl TE <dd>-libraries in 100 µl TE </body> </html>