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

Unknown Genomic Sequences 379
3. Methods
3.1. Purification of Genomic DNA and Restriction (see Note 1)
1. Isolate nuclei from cells of interest by suitable methods (see Note 1) and spin them down
to obtain the nuclear pellet.
2. Resuspend the pellet in 1 mL of 0.5 M EDTA. Avoid harsh vortexing and vigorous pipetting
to prevent shearing (see Note 1).
3. Add 25 µL of RNase A and 25 µL of Sarkosyl, mix by inverting the tube, and incubate
for 3 h at 37°C on a rotating wheel.
4. Add 25 µL of proteinase K, mix by inverting the tube, and incubate overnight at 37°C
on a rotating wheel.
5. Add 1 mL of phenol and mix by inverting the tube several times. Spin to separate the
phases and collect the lower phase and interphase (the lower phase is the aqueous phase
owing to the high density of 0.5 M EDTA).
6. Repeat step 5, but do not take the interphase.
7. Add 1 mL of phenolchloroform and collect the upper phase (which is now the aqueous
phase).
8. Dialyze overnight (or longer) against TE, pH 7.5, with at least four changes of TE. Avoid
a large increase in volume by keeping the dialysis bag tight.
9. Precipitate DNA with one-tenth vol of 0.3 M Na acetate and 2.5 vol of cold absolute
ethanol.
10. Spin at 4°C to collect DNA pellet, wash with 80% ethanol, remove residual ethanol, but
do not dry too long since the DNA will be difficult to redissolve.
11. Dissolve DNA in TE, pH 7.5, and store at 4°C.
12. Restriction digest of genomic DNA: Digest 10 µg of DNA with 1 U/µg of restriction
enzyme according to the manufacturer’s recommendation (see Notes 2 and 4) for at least
3 h (up to overnight).
13. Extract DNA once with phenol, once with phenol:chloroform, once with chloroform, and
precipitate with one-tenth vol of 0.3 M Na acetate and 2.5 vol of cold 100% ethanol.
14. Spin at 4°C, wash DNA pellet with 80% ethanol, and remove residual ethanol in the
SpeedVac. Again, do not dry too long. Resuspend DNA in TE, pH 7.5, and adjust
concentration to 1 µg/µL (OD at Aµ260).
15. Extract once more with chloroform, and remove traces of chloroform in the SpeedVac.
3.2. Purification of Oligonucleotide Primers and Annealing
of the Linker Fragment
1. Dry down 75 nmol of the oligonucleotide in the SpeedVac concentrator and dissolve in
75 µL of oligo loading mix. Heat for 5 min at 75°C and load 5∞15 µL onto a prerun
denaturing polyacrylamide gel. In a separate slot load some formamide-loading buffer
to monitor the run. Electrophorese until the bromophenol marker dye migrates to two
thirds of the gel.
2. By ultraviolet shadowing (6), locate the band corresponding to the full-length oligonucleotide
and excise from the gel using a razor blade.
3. Transfer the polyacrylamide gel slice to a 1.5-mL reaction tube containing 1 mL of PE
buffer and incubate overnight at 37°C.
4. Filter the supernatant through a 0.22-µm filter, prewetted with PE, with the help of a
2-mL syringe.
5. Wash another 100 µL of PE buffer through the filter.
6. Extract the pooled PE solutions with chloroform and dispense 2∞450 µL of the upper
phase into fresh tubes.