172
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
• For centrifuge columns: Sephadex G50 (medium), 1 ml syringes, glass
wool
• For minigels: agarose, TBE loading buffer (0.089 M Tris-borate, 0.089 M
boric acid, 0.002 M EDTA) suitable (1kb) size standard
Supplies • Water bath, 15°C
• Horizontal agarose gel electrophoresis apparatus
Procedure
Biotinylation of DNA probes by Nick translation
This method describes the labelling of2jlg probe DNA in a 100 J.!l reaction
volume.
Biotinylation 1. Combine: 2jlg of probe DNA, lOJ.!l of 10x reaction buffer, lOJ.!l of ß-mercaptoethanol, lOJ.!l of nucleotide stock, 20 units ofDNA polymerase I and
the tested volume of a 1:1000 dilution of DNase I, adjust to 100 J.!l with
double distilled water (enzymes should be added last).
2. Incubate for 2 hours at 15°C.
3. Place the reaction mixture on ice. It should be kept on ice until the actual
size of the reaction product has been determined.
4. Check the length of the probe molecules by gel electrophoresis: Remove
10 J.!l of the reaction mixture and add gelloading buffer. Denature by
boiling in a waterbath for 2-3 minutes. After another 3 minutes on
ice, the sample is loaded on a standard 1-2 o/o agarose minigel with a suitable size marker and run at 15 V/ern for 30 min. For visualization of
DNA, stain the gel in 0.5 f..Lgfml ethidium bromide and photograph under
UV -illumination.
5. For optimum hybridization conditions, the probe (which is visible as a
smear) should be 100- 500 nt in length. Depending on the result ofthe
electrophoresis proceed as follows:
a. If the probe size is within the desired range proceed to step 6.
b. If the probe size is too large, add more DNase I, incubate at 15°C
(usually higher amounts ofDNase I are added foranother 30 minutes),
repeat step 4.
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
• For centrifuge columns: Sephadex G50 (medium), 1 ml syringes, glass
wool
• For minigels: agarose, TBE loading buffer (0.089 M Tris-borate, 0.089 M
boric acid, 0.002 M EDTA) suitable (1kb) size standard
Supplies • Water bath, 15°C
• Horizontal agarose gel electrophoresis apparatus
Procedure
Biotinylation of DNA probes by Nick translation
This method describes the labelling of2jlg probe DNA in a 100 J.!l reaction
volume.
Biotinylation 1. Combine: 2jlg of probe DNA, lOJ.!l of 10x reaction buffer, lOJ.!l of ß-mercaptoethanol, lOJ.!l of nucleotide stock, 20 units ofDNA polymerase I and
the tested volume of a 1:1000 dilution of DNase I, adjust to 100 J.!l with
double distilled water (enzymes should be added last).
2. Incubate for 2 hours at 15°C.
3. Place the reaction mixture on ice. It should be kept on ice until the actual
size of the reaction product has been determined.
4. Check the length of the probe molecules by gel electrophoresis: Remove
10 J.!l of the reaction mixture and add gelloading buffer. Denature by
boiling in a waterbath for 2-3 minutes. After another 3 minutes on
ice, the sample is loaded on a standard 1-2 o/o agarose minigel with a suitable size marker and run at 15 V/ern for 30 min. For visualization of
DNA, stain the gel in 0.5 f..Lgfml ethidium bromide and photograph under
UV -illumination.
5. For optimum hybridization conditions, the probe (which is visible as a
smear) should be 100- 500 nt in length. Depending on the result ofthe
electrophoresis proceed as follows:
a. If the probe size is within the desired range proceed to step 6.
b. If the probe size is too large, add more DNase I, incubate at 15°C
(usually higher amounts ofDNase I are added foranother 30 minutes),
repeat step 4.