Bio-Rad CHEF Mapper® XA System - Manual (Page 57)

9.5 Pulsed Field Conditions by Organism
The table below shows the run parameters for various types of DNA samples. Photos and bar
codes for these examples are given in Section 9.7. All are performed at 14 °C except where noted.
DNA Agarose Pulse time Run time
DNA size (kb) conc. (seconds) (hours) Voltage Angle Buffer
Restriction 0.2–23 1.2% 0.09 a 3 9 V/cm 120° 0.5x TAE
fragments
5 kb ladder 5–75 1.0% 1–6 b 11 6 V/cm 120° 0.5x TBE
Lambda 50–1,000 1.0% 50–90 c 22 6 V/cm 120° 0.5x TBE
Ladder
Saccharomyces 200–2,200 1.0% 60–120 d 24 6 V/cm 120° 0.5x TBE
cerevisiae
Candida 1,000–4,000 0.8% 120 e 24 4.5 V/cm 120° 0.5x TBE
albicans 240 36
Schizo- 3,500–5,700 0.8% f 1,200 72 1.5 V/cm 120° 0.5x TAE
saccharomyces 1,800
pombe
Dictyostelium 3,600–9,000 0.8% f 2,000–7,000 h 158 1.8 V/cm 120° TBE i
discoideum 7,000–9,600 82 1.5 V/cm 120°
(a) 0.09 second single switch time for 3 hours.
(b) Ramped from 1 to 6 seconds over 11 hours.
(c) Ramped from 50 to 90 seconds over 22 hours.
(d) Ramped from 60 to 120 seconds over 24 hours.
(e) 120 second switch time for 24 hours followed by a 240 second switch time for 36 hours
(2 blocks).
(f) Chromosomal Grade Agarose
(g) Ramped from 20 minutes to 30 minute over 72 hours.
(h) Two blocks, with voltage change in second block. Run temperature 10 °C. Cox, et. al.,
Proc. Natl. Acad. Sci. USA, 87, 8247-8251 (1990).
(i) 27 mm Tris base, 27 mm boric acid, 0.75 mm EDTA, pH 8.5.
9.6 Blotting Megabase DNAs †
Southern Blot Transfer
Pulsed field electrophoresis is a powerful technique for physical mapping of genes in
various organisms. To determine the chromosomal location of a gene in a micro-organism
or the size of the restriction fragment containing a gene in mammalian systems, large DNA
fragments separated by CHEF are transferred onto membranes and detected by Southern
hybridization analysis. The procedures described for Southern transfer of DNA from stan-
dard agarose gels onto membranes are applicable to large DNA fragments separated by CHEF,
with the addition of the gel pretreatment step listed below.
Gel Pretreatment
Since DNA fragments larger than 20 kb cannot be transferred efficiently, DNA fragments
separated by pulsed field gels must be cleaved before transfer onto membranes. DNA can be
cleaved by using either acid (depurination) or UV irradiation. The depurination reaction is
† Contributed by Dr. Eric Lai, University of North Carolina
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