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HEINZ-ULRICH G. WEIER
2. Silane modification
l. Immerse precleaned dry slides in a solution of 0.1 % APS in 95% ethanol for 10 min.
2. Remove slides from the silane solution.
3. Rinse several times with water, and immerse in ultrapure water for
2 min.
4. Dehydrate by immersing in absolute ethanol.
5. Dry upright for 10 min at 65°C on a hot plate.
6. Store slides for 2-6 weeks at 4°C in a sealed box under nitrogen prior
to use.
Coverslip silanation is performed similar to the procedure described for
slides. Briefly, coverslips are rinsed with distilled water and dehydrated in
100% ethanol. Coverslips are derivatized with a 0.05-0.1 % solution of APS
in 95% ethanol for 2 min. Coverslips are then rinsed and dried as described
above (steps 3-4).
Subprotocol 2
Preparation of high molecular weight (HMW) DNA
The YAC clones used in the examples presented here, were part of the
CEPH/Genethon library, which is commercially distributed by Research
Genetics. Other clones might be used in their place without changing
the protocols. Information for many CEPH of the clones including insert
size, STS contents and radiation hybrid or genetic map position is available from the CEPH/Genethon server at URL http://www.genethonJr/
genethon_en.htmll or the Massachusetts Institute of Technology (MIT)
server (URL http://www-genome.wi.mit.edul).
The agarose plug preparation and pulsed field gel electrophoresis using
a CHEF electrophoresis system (BioRad) follow standard protocols. Typically, 5 to 15 individual YAC colonies are tested to account for deletions.
In most cases, the largest clone carries the least deletion(s).
The Pl/PAC/BAC clones typically show far fewer deletions, so that it
often suffices to pick 2-3 colonies from a plate, grow the cells overnight,
and extract the DNA using an alkaline lysis protocol. The DNA can then be
loaded directly onto the PFGE gel using a common gel loading dye.
HEINZ-ULRICH G. WEIER
2. Silane modification
l. Immerse precleaned dry slides in a solution of 0.1 % APS in 95% ethanol for 10 min.
2. Remove slides from the silane solution.
3. Rinse several times with water, and immerse in ultrapure water for
2 min.
4. Dehydrate by immersing in absolute ethanol.
5. Dry upright for 10 min at 65°C on a hot plate.
6. Store slides for 2-6 weeks at 4°C in a sealed box under nitrogen prior
to use.
Coverslip silanation is performed similar to the procedure described for
slides. Briefly, coverslips are rinsed with distilled water and dehydrated in
100% ethanol. Coverslips are derivatized with a 0.05-0.1 % solution of APS
in 95% ethanol for 2 min. Coverslips are then rinsed and dried as described
above (steps 3-4).
Subprotocol 2
Preparation of high molecular weight (HMW) DNA
The YAC clones used in the examples presented here, were part of the
CEPH/Genethon library, which is commercially distributed by Research
Genetics. Other clones might be used in their place without changing
the protocols. Information for many CEPH of the clones including insert
size, STS contents and radiation hybrid or genetic map position is available from the CEPH/Genethon server at URL http://www.genethonJr/
genethon_en.htmll or the Massachusetts Institute of Technology (MIT)
server (URL http://www-genome.wi.mit.edul).
The agarose plug preparation and pulsed field gel electrophoresis using
a CHEF electrophoresis system (BioRad) follow standard protocols. Typically, 5 to 15 individual YAC colonies are tested to account for deletions.
In most cases, the largest clone carries the least deletion(s).
The Pl/PAC/BAC clones typically show far fewer deletions, so that it
often suffices to pick 2-3 colonies from a plate, grow the cells overnight,
and extract the DNA using an alkaline lysis protocol. The DNA can then be
loaded directly onto the PFGE gel using a common gel loading dye.