8 Pulsed-Field Gel Electrophoresis: Protocols
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3. Seal the slot which contain blocks with 1 o/o liquified LMP agarose gel in
0.25 x TBE.
4. In case of bubbles, remove the air with a syringe.
5. Once the sealing LMP agarose has solidified ( -10 min), place gel into
chamber and cover with running buffer.
6. Set the appropriate voltage and switching times (see Table 1) and start
the run. Current (in Amps) should be approximately equal in the two
directions.
7. Remove gel after the run and stain in 0.25 x TBE containing EtBr ( 0.4 ~g/
ml).
8. Drain buffer from box with a pump and wash box with distilled water.
9. Photograph gel; DNA will get nicked during the exposure.
10. Rinse gel30 min in 0.25 M HCl to depurinate DNA and facilitate transfer.
11. Denature gel2 x 20 min in alkali ( denaturing solution), followed by 1-5
min in neutralization solution.
12. Transfer DNA to nylon membrane using standard Southern blotting
protocol. In general, blot PFGE gels Ionger than ordinary gels ( 48 h).
Table 1. Running conditions ofpulsed-field gel electrophoresis. OFAGE analysis using
the LKB-Pulsaphor Electrophoresis Unit. Specific pulse tim es select which molecules will
be resolved (separation range) and which remain unresolved (limiting mobility of the
gel). A more homogeneous resolution over the whole separation range can be achieved
by increasing switching times continuously (lower part of table).
Pulse Time(s)
Run Time (h)
Voltage {V)
Separation Range
(kb)
1
20
150
0- 50
5
20
150
10- 100
20
40
150
10- 350
40
40
150
30- 600
60
40
150
50- 800
80
40
150
50- 1000
100
40
150
50 - 1500