Pulsed Field Gel Electrophoresis
ProtocolBulletin 6227

Image Analysis
Imaging systems detect images, document and analyze
stained gels, and produce publication-quality outputs.
This protocol includes guidelines about gel staining andimage acquisition for pulsed field gel electrophoresis (PFGE).
Gel Staining
Process

1.	Stain the gel in ethidium bromide solution (40 µl of10 mg/ml stock solution per 400 ml) on a rocker orshaker for 25–30 min.
2.	Remove the gel to a new container and destain with~500 ml water.
3.	Change the water every 15–20 min — three 20-minwashes are recommended. It is important to use fresh
water for each wash.

Gel showing high background due to insufficient destaining.

4.	When stored properly, the staining solution can be used
for 10–20 gels. As it ages, staining fades and backgroundmay increase.
Note: Ethidium bromide is mutagenic and must be
disposed of properly according to institutional guidelines.
Nonmutagenic stains are available and may be used withPFGE gels after validation.

Gel showing low background following destaining.

Potential Problems

Aloe or powder from treated gloves leaves a residue on thegel which is visible on the image.
Recommendation

Avoid using gloves that are treated with either aloe or powder.
Untreated latex or nitrile gloves are acceptable.

Image Acquisition
Process

1.	Center the gel and zoom as close as possible to eliminate
blank space. Include wells and the bottom of the gel.
Do not include the space above wells and do not cut
off after the smallest band of the standard.

Do not take multiple images of the same gel.

Gel showing cloudiness and darkened areas due to handling the gel
while wearing nitrile gloves treated with aloe.

Zoom in to eliminate space around the gel.

Gel showing appropriate image acquisition.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6227

2.	Note that the ability to focus may be automatic or manual
depending on the imaging system used.
Tip: To make sure that the camera is in focus, use afluorescent ruler to focus on lines so that they are sharp;lines will appear highly pixilated (red).

3.	Avoid overexposure (overintegration) of the image.
This increases background and makes discriminatingclosely migrating bands difficult. In a correctly imagedgel, background is low and closely migrating bands aredistinct. Areas on the gel may appear slightly faint to thenaked eye, but will be visible when analyzing the TIFF withBioNumerics software.
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If available, select Highlight Saturated Pixels.
Saturated areas will appear red djust the integration time or the aperture untilA
all red is removedIf the image capture software allows it, collect numerous
images over a range of exposure times (for example,5 –25 sec)
Some saturation in the wells is acceptable

Example of a fluorescent ruler.

Gel image is overexposed.

Unfocused gel image.

Focused gel image.

Gel image is correctly exposed.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6227

4. Exposure time:■■■■If exposure time is too short, it is difficult to visualize faintbands at the bottom of the gel imageIf exposure time is too long, background is increased,
resulting in loss of distinction between closely
migrating bands
Underexposed image (6 sec)

Appropriate exposure (24 sec)

Overexposed image (60 sec)

Effect of increasing exposure time on a gel image.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6227

5. File Conversion■■■■

 ile conversion is not necessary for the Molecular Imager®
FGelDoc™ XR+ system with Image Lab™ software.
Select Export to PulseNet in the File menu ile conversion steps for GelDoc EQ and XR systems
Fand Molecular Imager® VersaDoc™ MP systems withQuantity One® software (version 4.5.0 or higher)
are illustrated below

Enter 113 toconvert the filesize to ~300 KB.

Export window in the Quantity One software.

Selecting “export view excluding overlays”
under Publishing in the Export window.

Entering 113 in the Specify field under
Resolution in the Export window produces
a file size ~300 KB.

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Life ScienceGroup

Bulletin 6227 Rev A

US/EG11-08641111

Sig 1211