Conditions for Separating DNAs of Various Sizes in CHEF Gels Agarose Size Range Switch TimesVCM Time

(kb)

(Hours)

1% Fast Lane

6-500

ramped, 3-80 seconds

6

18

10-800

ramped, 6-80 seconds

6

20

100-1000

60 seconds,

6

15

then 90 seconds

6

9

0.8% Fast Lane

150-2000

ramped, 30-180 seconds

5

24

All gels are run in 0.5x TBE. When very high resolution is required in the 800-2000-kb range, a lower voltage and a longer run time than those indicated above are used. If the gels are cast with Seakem GTG agarose, add 10% to the electrophoresis time indicated.

8. After electrophoresis, stain the gel in a 1 pg/ml solution of ethidium bromide or an appropriate dilution of SYBR Gold for 30 minutes at room temperature. Destain the gel in H2O for 30 minutes and photograph the gel under UV light.

9. After photography, incubate the gel in 250 ml of denaturation solution with gentle shaking for 30 minutes. Change the denaturation solution and incubate for an additional 30 minutes.

10. Transfer the DNA directly to a nylon membrane by capillary blotting in denaturation solution (for details, please see Chapter 6, Protocol 8).

Enhanced transfer of larger DNA fragments has also been noted when 6x SSC buffer is used in capillary blotting rather than the more standard 10x SSC solution.

11. After transfer, affix the DNA to the nylon membrane by baking for 2 hours at 80°C, by UV cross-linking, or by microwaving.

12. Carry out prehybridization and hybridization with labeled probes in a formamide-containing buffer (for details, please see Chapter 6, Protocol 10 ).

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