Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 27)

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.
† Contributed by Dr. Eric Lai, University of North Carolina
Discussion
1. The procedure is for 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 trans-
ferred 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 trans-
fer 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 sol-
vent with standard alkaline denaturation followed by neutralization. Alkaline transfer onto nylon
membranes gives as good or better sensitivity as standard transfers onto nitrocellulose 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 III or CHEF Mapper system can also be vacuum trans-
ferred onto nylon membranes in 4 hours using a commercial vacuum blotter, such as the
Model 785 Vacuum Blotter (catalog number 165-5000), and NaOH as buffer.
6. The DNA is transferred from the back of the gel (the side opposite the wells) onto the
membrane because irregularities in the surface of the gel frequently occur during solidi-
fication of these high percentage gels (1%). These surface artifacts will interfere with the
transfer of the DNAs from the gel. Transfer from the other side of the gel insures smooth
surface contact between the gel and the membrane.
7. It is essential to neutralize the membrane after transfer to prevent changing the pH of the
hybridization buffer during hybridization.
8. It is not absolutely necessary to bake nylon membranes after alkaline transfer since the
DNA should be fixed onto the membrane by NaOH.
9. To monitor the efficiency of the transfer, stain the gel in neutralization buffer for 30 minutes
with 1 µg/ml EtBr. Take a photograph of the post-transferred gel, and compare with the
original picture.
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