18 Quantitative DNA Fiber Mapping
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- RNase (Roche Molecular Biochemicals), DNase-free: boil at 100°C for
10 min., aliquot and store at -20°C
- Salmon sperm DNA (3'-5', Boulder, CO), 20 mglml. Store at -20°C.
- Sodium dodecyl sulfate (SDS)(Na salt, Sigma Chemicals): 10% in water.
- Thermus aquaticus (Tag) DNA polymerase,S U/IlI (Perkin Elmer).
Store at -20°C.
- Ultrapure water (Mallinckrodt, cat. # H453)
- Yeast artificial chromosome (YAC) library (Research Genetics). Store
at -80°C.
- YOYO-l (Molecular Probes): Stock is ImM in DMSO. Dilute 1:1000
with water prior to use. Store at -20°C and discard diluted dye after
1 week.
- Zymolase (Sigma Chemicals, 70,000 U/g): prepare 10 mg/ml in 50 mM
KH z P0 4 , pH 7.8, 50% glycerol. Store at -20°C.
- AHC medium (BIO 101): add 36.7 g ofAHC powder per liter of purified
water, autoclave at 121°C for 15 min.
- AHC agar (BIO 101): add 53.7 g of AHC agar medium per liter of purified water. Autoclave at 121°C for 15 min. Cool to 50°C. Then, mix well
and pour plates. Store plates at 4°C.
- Alkaline Lysis (AL) solutions sufficient for 12 preps at the level of20 ml
cell culture:
- AL Solution I: 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCI pH
8.0, add 4 rnl of 0.5 M glucose, 0.8 ml of 0.5 M EDTA and 1 ml of 1 M
Tris-HCI to 34.2 ml water. Store at 4°C.
- AL Solution II: 0.2 N NaOH, 1% SDS. Add 1.4 ml of 10 N NaOH, 7 ml
of 10% SDS to 61.6 ml water.
- AL Solution III: 3 M NaOAc, pH 4.8
- Antifade solution: 1% p-phenylenediamine (Sigma), 15 mM NaCl,
1 mM H Z P0 4 , pH 8.0,90% glycerol. Store at -80°C.
- Blocking stock solution: dissolve blocking reagent (Roche Molecular
Biochemicals, cat. # 1096 176) in maleic acid buffer (10% w/v) with
shaking and heating. Autoclave stock solution and store in aliquots
at 4°C.
Buffers and other
solutions