Bio-Rad CHEF-DR II System - Manual (Page 30)

6. Carefully remove the paper towel and blotting papers. Remove the membrane together
with the gel, turn over the membrane and gel, lay them gel side up, and mark the location
of the wells and the orientation marker on the top of the gel. The position of the wells
can be accurately marked on the membrane by using a fine point permanent marker pen,
cutting through the bottoms of the wells.
7. Neutralize the membrane in 0.5M Tris, pH 7.0 (neutralization buffer) for 5 minutes, fol-
lowed by rinsing briefly in 2x SSC. Transferred DNA can be visualized on the mem-
brane by placing the damp blot on a transilluminator.
8. Dry the membrane by blotting onto 3MM or other adsorbent paper and proceed with
hybridization. UV crosslinking of the DNA to the membrane is not recommended with
this alkaline transfer method.
Discussion
1. The procedure is based on gels approximately 6 mm thick. If thicker gels are used, the
staining period may be prolonged to allow diffusion of EtBr into the middle of the gels.
DNA that is not stained with EtBr will not be nicked by the UV light and thus will not be
transferred from the gel.
2. If the output of the UV light source is not known and no UV meter is available, you can
titrate your UV light source as follows. Run a CHEF gel with eight lanes of S. cerevisiae
chromosomes as markers using a switch time that will provide resolution from 200-1,000
kb. Stain the gel with EtBr, and photograph with medium-wave 302 nm UV light and
fast film (Polaroid type 667) to minimize nicking of DNA. Note the exposure time of the
photo. Cut the gel into eight strips, each containing a lane of separated yeast chromo-
somes. UV irradiate the strips with a 254 nm light source for time intervals of 5, 10, 15,
30, 45, 60, 90, and 300 seconds. If a 254 nm light source is not available, 302 nm light can
be used, but exposure times have to be lengthened approximately five-fold. Alkaline
transfer the gel strips as described, and stain the gels after transfer. Take a photograph of
the gel strips using the same UV light source, film, and exposure time as before transfer,
and compare it with the photograph before transfer. Choose the time period that results in
80-90% transfer of DNA. Do not choose the time intervals with complete transfer because
most of the transferred DNA fragments will be too short for effective hybridization. If
less than 10 second short-wave UV irradiation is required, you may need to use a 302
nm light source for taking the picture of the gel and cutting away excess gel area. As a gen-
eral rule, 10 seconds or less exposure time is needed with a new UV transilluminator.
The UV output will decrease with time, to as little as 30% of its initial rating after 7 years.
3. Presoaking the gel in NaOH prior to transfer decreases background and increases trans-
fer efficiency.
4. Pulsed field gels can also be blotted onto membranes using 20x SSC as the transfer buffer
solvent with standard alkaline denaturation followed by neutralization. Alkaline transfer
onto nylon membranes gives as good or better sensitivity as standard transfers onto nitro-
cellulose filters. The alkaline procedure is much simpler and faster. In addition, nylon
membranes can be reused many more times than nitrocellulose filters. Some blots may be
reused as many as 20 times.
5. DNAs separated on the CHEF-DR II, CHEF-DR III or CHEF Mapper ® system can also
be vacuum transferred onto nylon membranes in 4 hours using a commercial vacuum
blotter, such as the Model 785 Vacuum Blotter (165-5000), and NaOH as buffer.
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