8 Pulsed-Field Gel Electrophoresis: Protocols
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2. Mix with an equal volume of 2% LMP agarase (kept at 45°C) in TE.
3. Pipet mixture into precooled block formers.
4. Incubate in TE+ 100 mM NaCl for 2 days at room temperature
Yeast chromosomes
See chapter on YACs
See chapter on Y ACs
Pre-cool block formers on ice
• Day 1: <1 h
• Day 2: 2 h
• Day 4: ready for use
Procedure
1. Picksingle colony from a YPD plate into 10 ml YPD preculture broth;
grow with vigoraus shaking 24h at 30°C. Then add 200 ml YPD broth and
shake for 24-48 h (yields approx. 100 blocks).
2. Spin 4K/10 min, then resuspend in 50 mM EDTA/10 mM Tris-HCl (pH
7.5).
3. Spin4K/10 min and resuspendin SCE (1M sorbitol + 0.1 M Na citrate, pH
5.8 + 10 mM EDTA (pH 7.5)).
4. Count the cells in aN eubauer chamber by diluting an aliquot of the cell
suspens10n.
5. Cells are resuspended (after centrifugation) in SCE suchthat 5 x 10 7 cells
are contained in 40 111 SCE (corresponds to 5 x 10 7 cells/block of 80 11l).
6. The solution is mixed with 0.1 mg/ml Zymolyase 100T and 100 mM bmercaptoethanol and kept at 37°C for 15-30 min.
7. Mix an equal volume ofthe cell suspension with 2% LMP agarase (kept at
50°C) in SCE.
Reagents
Salutions
ln advance
Time required