John M. S. Bartlett.pdf - Bio-Nica.info

380 Quivy and Becker
7. Precipitate the oligonucleotides by the addition of 36 µL of 5 M LiCl, 4.5 µL of 1 M
MgCl 2 , and 1 mL cold 100% ethanol.
8. Mix by vortexing and let precipitate at –70°C for 15 min.
9. Spin at 4°C for 20 min in a tabletop centrifuge, wash the pellet with 80% ethanol, dry in
the SpeedVac, and resuspend the oligonucleotide in TE, pH 7.5.
10. Determine the concentration (OD at A 260 ) and dilute some aliquots to the working
concentration.
11. Annealing of linker oligos: combine in a 1.5-mL reaction tube: 20 pmol/µL of each linker
oligonucleotide in 250 mM Tris (pH 7.5), 5 mM MgCl 2 . Heat at 95°C for 5 min, transfer
to a beaker containing boiling water, and allow to cool slowly at room temperature for 5 h
(up to overnight in the cold room). Aliquot the linker solution and store at –20°C. Aliquots
are only used once and never refrozen.
3.3. Kinasing of Primer 3
1. Combine in a tube 1 µL of P3 (10 pmol), 3 µL of 10× T4 polynucleotide kinase, and
10.5 µL of water.
2. Add 15 µL of γ 32 PATP and 0.5 µL of polynucleotide kinase.
3. Incubate for 30 min at 37°C.
4. Add 20 µL of 50 mM EDTA, pH 8.0, and heat at 65°C for 10 min.
5. Purify the labeled oligonucleotide from the unincorporated label by a G-25 spin column.
3.4. First Primer Extension
1. Combine in a tube: 0.5 to 1 µg of restricted genomic DNA (see Subheading 3.1. and Note 4),
1 µL P1 (0.5 pmol), 1 µL 1 N NaOH, and water to 8 µL.
2. Incubate at 65°C for 5 min.
3. Immediately add 2 µL of TES buffer and mix.
4. Spin to collect and incubate for 10 min at room temperature.
5. Add 9 µL of a mix containing 2 µL of 10× Vent buffer, 0.4 µL of 10 mM dNTPs,
6.6 µL of H 2 O.
6. Incubate at 50°C for 10 to 20 min.
7. Add 1 µL of the Vent Exo (2 U).
8. Incubate for 10 min at 76°C.
9. Chill on ice, spin to collect liquid, and proceed immediately to ligation.
3.5. Ligation
1. Prepare a premix containing 5 µL of 10× ligase buffer, 19 µL of 40% PEG 8000, and
5 µL of annealed linker (20 pmol/µL). Mix well by pipetting since the resulting solution
is very viscous.
2. Add 29 µL of this premix to the first primer extension reaction (see Section 3.4.).
3. Add 1 µL of T4 DNA ligase (400 U), mix well by pipetting, and incubate overnight
at 17°C.
3.6. PCR
1. To the ligation reaction, add 150 µL of TE, pH 8.5, and mix by vortexing.
2. Add 150 µL of phenolchloroformisoamyl alcohol (25241) mix by vortexing, and
then spin for 5 min.
3. Collect the upper aqueous phase and transfer to a tube containing 10 µL of 7.5 M
NH 4 Ac/yeast tRNA. Add 750 µL of cold 100% ethanol, mix by vortexing, and let
precipitate for 15 min at –20°C.