Spring 2024

BMS 340 – Assignment #2

Pulsed- Field Gel Electrophoresis (PFGE)

Question 1
The following cases and photos are potential problems that may have occurred
during the operation of pulsed- field gel electrophoresis (PFGE). Observe each case
and write your recommendations to solve these problems.

1- DNA fragments do not run evenly and spots or debris appear on gel images.

-There are several issues that could cause spots or speckles on

the gel image.

-Recommendations:

1-Make sure the agarose is completely melted by heating it in

a microwave and pulse it briefly, being careful not to boil it over.

2- Throw away expired agarose.

3- Monitor lot numbers and discard deteriorated lots that may be associated with
subpar outcomes.

4-Only use clean glassware, combs, and gel forms.

5- Use premium water to make the 0.5x TBE buffer that is used for the gel and
running buffer.

6- Before the gel solidifies, remove any visible particles, dust, and lint.
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2- Buffer flow is uneven or interrupted leading to curved or slanted Lanes

Recommendations:

1- To prevent the agarose from leaking, make sure the frame

is sealed and the screws are tightened before adding gel.

2-Turn the connectors around and clear the obstructions in the lines.

3- Before beginning a run, balance the electrophoresis chamber.

4- Make sure the pump is operating and that the flow rate (1 L/min) is appropriate.

3- Problems with the adjusted and applied voltage gradient

-Band resolution and band appearance in gels

are also impacted by the voltage gradient, which

needs to be adjusted as necessary.

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1-The bands are compressed.

2-appropriate band patterns

3-bands run off the gel

4- Incorrect band separation

-short and appropriate electrophoresis run lengths

A- The doublet was not resolved in the gel and the run time was insufficient

B- The doublet in the gel was resolved when the run time was extended.

-Recommendations: Prevent variables like the composition of the 0.5× TBE buffer,
using commercial versus homemade buffers, buffer pH, and buffer temperature
that can impact run times.
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5- The chamber or the tubing is contaminated, affecting the entire gel.

Additionally, a single tube or reagent may be contaminated, affecting only
one or several lanes. As a result, DNA will be separated as a smear on a gel.
Recommendations:
1-Between runs, keep the chamber dry and clean.
2- Run two liters of 5-to 10% bleach through the electrophoresis chambers to clean
them, tubing and chamber for half an hour. Turn off the chiller.
3- Use already tested PFGE plugs to run the gel.
4- If required, repeat the cleaning.

References:

Herschleb, J., Ananiev, G., & Schwartz, D. C. (2007). Pulsed-field gel electrophoresis. Nature
protocols, 2(3), 677-684.

Kaufmann, M. E. (1998). Pulsed-field gel electrophoresis. Molecular bacteriology: protocols

and clinical applications, 33-50.