Troubleshooting - Hoefer HE33 User Manual

problem
Deformed sample well
Samples not running along a straight path
Double-banded pattern
Poor band resolution
Foam pads peel off
p12
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Troubleshooting

solution
Allow the gel to set for a minimum of 30 minutes and make
sure it is at room temperature before removing the comb.
When removing the comb, hold it at a slight angle and lift
very slowly to prevent the gel from breaking.
Take care to not damage the well with the pipet while loading
the sample; aim for the center of the well and do not punc-
ture the bottom with the pipet tip.
If a comb or running tray is warped, replace.
Reduce the voltage.
Choose a buffer with the appropriate ionic strength and buff-
ering capacity. (The buffering capacity of TBE, for example,
is higher than that of TAE.) If the buffer is depleted, stop the
run, remove the lid, and pipette the buffer from each cham-
ber into the opposite chamber to replenish the buffer.
If the gel is uneven, level the casting tray before pouring the gel.
The comb must be vertical to prevent well shape distortion.
Decrease the buffer level to 1 mm above the top of the gel,
to reduce the vertical temperature gradient.
Add Ficoll ™ ™ ™ , glycerol, or sucrose to the sample loading buffer , glycerol, or sucrose to the sample loading buffer
to ensure that the sample sinks to the bottom of the well.
(Ficoll is preferred.)
Make sure the sample is completely dissolved.
Reduce the voltage.
Reduce the sample concentration.
Reduce the sample volume.
At least 1 mm of gel below the bottom of the comb is required
to prevent samples from leaking out of the well bottom.
Reduce the salt concentration of the sample.
Check enzyme activity; the sample may require longer
digestion or a different restriction buffer.
Prepare fresh sample if you suspect nuclease contamination.
Choose agarose with a low endosmosis value.
Install the running tray as as described on page 5; do not
press straight down into place.