Pulsed Field Gel Electrophoresis
ProtocolBulletin 6228

Pulsed Field Gel Electrophoresis Standards
Bio-Rad offers standards for all pulsed field gel electrophoresis(PFGE) applications, from field inversion gel electrophoresis(FIGE) separation of cosmid inserts to the largest
chromosomal separations. Higher molecular weightstandards are prepared in low-melt agarose blocks thatcan be cut to fit any well dimensions.
No more than four samples should be placed between lanes
with standards. Placement of standards may be adjusted
when fewer than 7 (for 10-well gels) or 11 (for 15-well gels)samples are loaded on a gel.

PulseNet Global Reference Standard:
Salmonella enterica, serotype Braenderup H9812
Information in this section is intended for PulseNet members.
H9812 is used as the global reference standard by PulseNet
member laboratories. It was selected because when digested
with XbaI, it has an optimal number of bands (18) distributed
across a wide range of sizes (20.5–1,135 kb).
■■■■

For 10-well gels, load standards in lanes 1, 5, and 10
For 15-well gels, load standards in lanes 1, 5, 10, and 15■■

 9812 is used to normalize migration variation occurring
Hacross the gel and to accurately determine sample band sizes 9812 is always digested with XbaI, regardless of whichH
enzyme is used to digest the actual samples
 o more than 4 samples should be placed between H9812
Nlanes. Placement of standards may be adjusted when fewer
than 7 (for 10-well gels) or 11 (for 15-well gels) samples areloaded on a gel:
— For 10-well gels, load standards in lanes 1, 5, and 10— For 15-well gels, load standards in lanes 1, 5, 10, and 15
1A

BCDE5F

Standard and markers. A, 5 kb ladder standard; B, 8.3–48.5 kb standard;C, lambda ladder standard; D, S. cerevisiae marker; E, H. wingei marker;
F, S. pombe marker.

Lanes 1, 5, and 10 include the H9812 standard.

10

Recommendations■■■■

 rior to using a new lot of H9812 plugs, compare it withPa previously used lot to confirm that the new lot produces
an acceptable PFGE pattern, that is no pattern changes,no changes in band intensity, and no DNA degradation
 retested H9812 standard plugs serve as a positiveP
control for XbaI digests and can be useful introubleshooting efforts:—	If a problem is observed in sample lanes but not thestandard lanes, it was caused by something that
affected only the sample (for example, sample plug
preparation, BlnI digest)—	If a problem is observed in both sample and standard
lanes, it is caused by a step involving both sample
and standard plugs (for example, XbaI digest,electrophoresis)

Tip: Make an H9812 plug when preparing sample plugs tocontrol for parameters and reagents used to make plugs onthat day. If H9812 and sample plugs have deteriorated butpretested H9812 standards appear appropriately, the problem
occurred during plug preparation.

S. enterica, serotype Braenderup H9812 standard.

Bio-RadLaboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
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Taiwan 886 2 2578 7189 Thailand 800 88 22 88 United Kingdom 020 8328 2000

Life ScienceGroup

Bulletin 6228 Rev A

US/EG

11-08641111

Sig 1211