18 Quantitative DNA Fiber Mapping
235
The DNA is recovered from the low melting point agarose slab gel by
excising the appropriate band using a knife or razor blade. High molecular
weight DNA is then isolated by ~-agarase digestion of the gel slices.
Procedure
1. Pulsed field gel electrophoresis (PFGE)
I. Spin down cells from 5 ml AHC media at 400 rpm for 6 min. Resuspend
cells in 0.5 ml of 0.125 M EDTA, pH 7.8. Spin again and remove supernatant.
2. Resuspend the - 70 III cell pellet in 500 JlI of SCE. Mix with an equal
volume of 1.5% LMP agarose preheated to 43°e. Quickly pipette up and
down, then vortex for 1-2 s to mix. Pipette into plug molds (Biorad)
and allow to solidify at room temperature or on ice.
3. Remove plugs from molds, incubate samples in 2 ml SCE containing
100 III of zymolase and shake at 150 rpm at 30°C for 2.5 h to overnight.
4. Remove SCE and add 2 ml of ES containing 100 III of proteinase K
(20 mg/ml). Shake 5 h to overnight at 50°e.
5. Remove ES and rinse 5 times with 6 ml of TE50 for 30 min each rinse.
Store the plugs at 4°e.
I. YACs: voltage gradient, 6 V/cm; switching time, 79 s initially, 94 s final;
running time, 38 h; agarose concentration, 1.0% LMP agarose; running
temperature, 14°C; running buffer, O.5xTBE.
2. Pl/PAC/BAC clones: voltage gradient, 6 V/cm; switching time, 2 s initially, 12 s fmal; running time, 18 h; agarose concentration, 1.0% LMP
agarose; running temperature, 14°C; running buffer, O.5xTBE.
For probe production and determination of optimal PFGE conditions:
stain the gel with ethidium bromide (EB, 0.5 Ilg/ml in water), cut out a
gel slice containing the target DNA band and transfer slice to a 14 ml polystyrene tube (CaL# AS-2264, Applied Scientific). Wash slice with ultrapure
water for 30 min, and then wash with 1x agarase buffer for 30 min.
For high molecular weight DNA isolation: run duplicate samples on the
right and left side of the gel, respectively. After a predetermined run time,
cut gel in half, and stain one half with EB. Measure the migrated distance
Preparation of gel
plugs containing
YACs
PFGE running
conditions
235
The DNA is recovered from the low melting point agarose slab gel by
excising the appropriate band using a knife or razor blade. High molecular
weight DNA is then isolated by ~-agarase digestion of the gel slices.
Procedure
1. Pulsed field gel electrophoresis (PFGE)
I. Spin down cells from 5 ml AHC media at 400 rpm for 6 min. Resuspend
cells in 0.5 ml of 0.125 M EDTA, pH 7.8. Spin again and remove supernatant.
2. Resuspend the - 70 III cell pellet in 500 JlI of SCE. Mix with an equal
volume of 1.5% LMP agarose preheated to 43°e. Quickly pipette up and
down, then vortex for 1-2 s to mix. Pipette into plug molds (Biorad)
and allow to solidify at room temperature or on ice.
3. Remove plugs from molds, incubate samples in 2 ml SCE containing
100 III of zymolase and shake at 150 rpm at 30°C for 2.5 h to overnight.
4. Remove SCE and add 2 ml of ES containing 100 III of proteinase K
(20 mg/ml). Shake 5 h to overnight at 50°e.
5. Remove ES and rinse 5 times with 6 ml of TE50 for 30 min each rinse.
Store the plugs at 4°e.
I. YACs: voltage gradient, 6 V/cm; switching time, 79 s initially, 94 s final;
running time, 38 h; agarose concentration, 1.0% LMP agarose; running
temperature, 14°C; running buffer, O.5xTBE.
2. Pl/PAC/BAC clones: voltage gradient, 6 V/cm; switching time, 2 s initially, 12 s fmal; running time, 18 h; agarose concentration, 1.0% LMP
agarose; running temperature, 14°C; running buffer, O.5xTBE.
For probe production and determination of optimal PFGE conditions:
stain the gel with ethidium bromide (EB, 0.5 Ilg/ml in water), cut out a
gel slice containing the target DNA band and transfer slice to a 14 ml polystyrene tube (CaL# AS-2264, Applied Scientific). Wash slice with ultrapure
water for 30 min, and then wash with 1x agarase buffer for 30 min.
For high molecular weight DNA isolation: run duplicate samples on the
right and left side of the gel, respectively. After a predetermined run time,
cut gel in half, and stain one half with EB. Measure the migrated distance
Preparation of gel
plugs containing
YACs
PFGE running
conditions