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

382 Quivy and Becker
7. Prepare a premix containing 0.2 pmol labeled P3, 1.5 µL of 10× circumvent sequencing
buffer, 1 µL of Triton X-100, and add water to 15 µL.
8. Concentrate beads and then remove supernatant.
9. Resuspend the beads in 15 µL of premix.
10. Transfer 3.5 µL of this bead suspension into each tube containing a termination mix and
mix by pipetting.
11. Heat at 95°C for 10 s and then transfer to 50°C. Incubate for 10 to 20 min with occasional
gentle resuspension.
12. Add 1 µL of Vent Exo (2 U) to each tube, mix well, and immediately transfer to 76°C.
13. Incubate for 10 min and then chill on ice.
14. Spin to collect liquid, concentrate beads, and discard supernatant.
15. Resuspend beads in 50 µL of water, spin shortly to collect liquid, and concentrate beads.
16. Carefully remove all liquid and resuspend the beads in 4 µL of formamide loading
buffer0.15 NaOH (21). Mix well to dissolve all aggregates (see Note 8).
17. Leave for 5 min at room temperature.
18. Incubate 3 min at 76°C, spin to collect liquid, and chill on ice.
19. Concentrate beads and then transfer supernatant into a fresh tube on ice.
20. Check with a hand monitor that most of the radioactivity is in the supernatant. The bead
pellet always contains radioactivity owing to trapped P3. Usually, we do not re-extract
the beads.
21. Load on a prerun sequencing gel (see Chapter 51); reactions can be stored at –20°C.
22. Fix gel in fixing solution, dry onto blotting paper, and expose the dried gel to X-ray film
in the presence of an intensifying screen. Readable sequences are usually obtained after
overnight exposure.
4. Notes
1. The genomic DNA used for genomic sequencing needs to be clean and undegraded. Any
shearing of the DNA during preparation and handling before the first primer extension must
be avoided. Nicks between the restriction site and the P1 priming site will be converted to
blunt ends during the first primer extension and will give rise to a background of dominant
bands in all four sequencing lanes. We detail here one particular protocol that consistently
yields high molecular weight genomic DNA of good quality. In principle, other methods
can be followed. In any case, we advise to start a DNA prep with a nuclei isolation. Some
suggestions for how to prepare nuclei have been described (7,8).
2. Enzymes that produce blunt ends, with 5′- or 3′-overhangs can be used because the
fragment is anyway converted to a blunt end after the first primer extension.
3. All oligonucleotides should be gel-purified (see Subheading 3.2.) and stored in water or
TE, pH 7.5, at two concentrations: a stock solution (determined after the purification) and
a diluted working solution that should not be frozen and thawed more than five times.
The sequence-specific primers 1–3 need to be designed for each new sequencing project.
The long and short linker oligonucleotides are constant. The distances between primers
1/2 and 2/3 should not be longer than 10 bases; overlaps of up to 5 bp are tolerated,
but not required.
4. This protocol was used to sequence in the context of the Drosophila genome, which
required changes from the original protocol described for mammalian DNA. If sequences
are to be determined in the context of the 10 times more complex mammalian genome,
a few parameters have to be adjusted: The amount of starting DNA used should be
increased about five times, and the longer-linker oligos should be taken from the original
procedure (3).