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JORN KOCH
chain elongation is simply replaced with a similar molar amount of the
corresponding dideoxy-analogue (Roche).
- Tth DNA polymerase and 10 x polymerase buffer: enzyme and buffer
are supplied together by Roche
- Stop buffer: 50 mM NaCI, 50 mM EDTA, pH 8.0.
- Washing buffer: 4xSSC, pH 7.0, 0.05% Tween 20. (l xSSC: 150 mM
NaCl, 15 mM sodium citrate)
- Blocking solution: 5% non-fat dry milk dissolved in washing buffer.
Centrifuge for 2 min in an Eppendorf centrifuge and use supernatant.
- Antifade solution: Vectashield from Vector Laboratories
- Counterstain: either propidium iodide (Sigma) or DAPI (Sigma)
- Either, two thermoblocks with a metal surface to ensure good heat
transfer, one with a surface temperature of 94°C and the other with
a surface temperature of 55-65°C. The temperature of the 94°C incubator should be scrupulously measured, since it must not deviate more
than I-2°C from the desired value. To ensure this, it is important that
the slide and the hot plate is covered with an insulating lid.
- Or a special PRINS/in situ peR machine (from Hybaid, MJ-Research
or Techne) preset (simulated slide function) to incubate the slide at 9194°C for 2-4 min and at 55-65°C for 5-60 min (depending on the oligonucleotide used). This is the more convenient set-up and ensures
absolute reproducibility of the reaction conditions.
Both types of incubators should be covered with a lid (if a lid is not
built in), to optimize temperature control.
- For a multicolor PRINS an ice-cold (-20°C) ethanol series (70%, 90%,
99%) is also needed.
Procedure
Standard PRINS
1. To cover a standard slide completely, prepare 50 III reaction mixture
containing: 38 JlI ddH 2 0, 5 JlllO x polymerase buffer,S JlII0xdNTP,
1 JlI DNA probe (from 500 ng and up) and 1 JlI DNA polymerase
(l unit) and mix gently by tapping a finger on the Eppendorf tube.
JORN KOCH
chain elongation is simply replaced with a similar molar amount of the
corresponding dideoxy-analogue (Roche).
- Tth DNA polymerase and 10 x polymerase buffer: enzyme and buffer
are supplied together by Roche
- Stop buffer: 50 mM NaCI, 50 mM EDTA, pH 8.0.
- Washing buffer: 4xSSC, pH 7.0, 0.05% Tween 20. (l xSSC: 150 mM
NaCl, 15 mM sodium citrate)
- Blocking solution: 5% non-fat dry milk dissolved in washing buffer.
Centrifuge for 2 min in an Eppendorf centrifuge and use supernatant.
- Antifade solution: Vectashield from Vector Laboratories
- Counterstain: either propidium iodide (Sigma) or DAPI (Sigma)
- Either, two thermoblocks with a metal surface to ensure good heat
transfer, one with a surface temperature of 94°C and the other with
a surface temperature of 55-65°C. The temperature of the 94°C incubator should be scrupulously measured, since it must not deviate more
than I-2°C from the desired value. To ensure this, it is important that
the slide and the hot plate is covered with an insulating lid.
- Or a special PRINS/in situ peR machine (from Hybaid, MJ-Research
or Techne) preset (simulated slide function) to incubate the slide at 9194°C for 2-4 min and at 55-65°C for 5-60 min (depending on the oligonucleotide used). This is the more convenient set-up and ensures
absolute reproducibility of the reaction conditions.
Both types of incubators should be covered with a lid (if a lid is not
built in), to optimize temperature control.
- For a multicolor PRINS an ice-cold (-20°C) ethanol series (70%, 90%,
99%) is also needed.
Procedure
Standard PRINS
1. To cover a standard slide completely, prepare 50 III reaction mixture
containing: 38 JlI ddH 2 0, 5 JlllO x polymerase buffer,S JlII0xdNTP,
1 JlI DNA probe (from 500 ng and up) and 1 JlI DNA polymerase
(l unit) and mix gently by tapping a finger on the Eppendorf tube.