Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine
http://hg.wustl.edu/hdk_lab_manual/yeast/yeast10.html
Purpose:
To identify the YAC subclones containing both a human insert and a portion of either the left or right arm of the pYAC4 vector. Identification of these clones is necessary in order to do YAC chromosome walking, and is also useful in the determination of whether a particular YAC clone has a contiguous human insert or whether a co-cloning event has occurred. Vector arm sequences are identified using pBR322 fragments from a BamHI-PvuII double digest. Note: A PCR procedure for finding endclones is now used in the lab. See J. Howe for the protocol.
Time required:
- Probe preparation takes 2 days
- Hybridization takes 1-2 days
Special Reagents:
Procedure:
- Digest 30 礸 pBR322 with 50 units BamHI in a 300 祃 volume (including 30 祃 10X BamHI buffer); incubating at 37 degrees C for 4 hours.
- Phenol extract the DNA. Follow with a chloroform extraction, then ethanol precipitate the DNA. Wash the DNA pellet with 70% ethanol. Dry the pellet, then resuspend in 270 祃 TE and 30 祃 10X PvuII buffer. Add 50 units PvuII, incubate at 37欳 for 4 hours. Heat to 70 degrees C for 15 minutes to inactivate the enzyme, then add 33 祃 10X glycerol stop mix.
- Set up a 0.8-1.0% TAE agarose gel ahead of time: tape 10 wells of a 30 well comb together to form a common well, then pour the gel. Load the entire digest into the large well, and load 1 kb ladder several wells away.
- Electrophorese at 60V for 14 hours using a pump to recirculate buffer. Take the gel to the UV prep box (use long wavelength UV) and cut out the 2.7 kb (left arm probe) and 1.7 kb (right arm probe) bands. Place gel slices into separate small dialysis tubing (10 mm) and add 1 ml TE. Fasten clips, then electroelute for 4-6 hours at 100V.
- Remove the gel slice from bag, then clip; rinse the inside of bag by running between gloved fingers. Collect fluid, then wash once more with 1 ml of TE. Collect as before, then extract with phenol. Chloroform extract and ethanol precipitate the DNA. Wash DNA pellet in 70% ethanol, then dry. Resuspend the DNA in 20 祃 TE, then determine concentration by running an aliquot on a mini-gel with concentration standards. Also use a size standard and check that the bands are the appropriate sizes.
- These pBR322 fragments can now be used in conventional hybridization procedures. YAC subclones which have strong signals after hybridization with one of these probes will represent clones which contain a portion of the appropriate pYAC4 vector arm. These fragments are also useful in pulsed-field mapping of YAC clones.
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