DNA Polymerase 21
- 0.5M EDTA, pH 8.0.
- TNE buffer:
- 10 mMTris-HCl, pH 8.0
- 100 mM NaC1
- 1 mM EDTA
- TE buffer:
- 10 mMTris-HC1, pH 8.0
- 1 mM EDTA
- Sephadex G-50 equilibrated in TNE buffer.
- o~-32P dATP (10 mCi/mL, 3000 Ci/mmol).
- Sterile distilled water.
2.3.1.2. EQUIPMENT REQUIRED - Water bath (15°C).
- Water bath (70°C) or dry beating block.
- Bench-top centrifuge.
- Sterile glass wool.
- Sterile 1.5-mL microfuge tubes.
- Pipets and sterile pipet tips.
- Ice bucket.
2.3.1.3. PROTOCOL
- In a sterile tube on ice mix the following:
- Probe DNA (up to 1 pg)
- 5 ~L Nick translation buffer
- 200 pM dGTP, dCTP, dTTP (5 I.tL each)
- 5 ~L a-32p dATP (50 gCi, 20 ~/)
- 2 ~L DNase I (200 pg)
- 1 gL DNA polymerase I (2 U)
- Sterile distilled water to 50 ~L
- Incubate at 15°C for 50 min.
- Stop the reaction by addition of 2 pL of 0.5M EDTA, pH 8.0, or by
heating to 70°C for l0 min. - Purify the labeled probe from unincorporated nucleotides by a spun
column method. Prepare a column of Sephadex G-50 in a 200-taL pipet
tip plugged with glass wool. Wash the column with 500 ~L TNE buffer,
by supporting it in a microfuge tube and spinning at 2000g for a few
seconds. Load the nick translation reaction onto the column, and spin
for 30 s. Elute the labeled probe with a futher 100 gl_, of TNE (or TE)
buffer and a further spin. Pool the run-through and eluate, and then
discard the column containing the unincorporated nucleotides.