Hoefer HE99X - Manual (Page 17)

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p9
Troubleshooting
problem solution
Sample well deformed Allow the gel to set for a minimum of 1 hour and make
sure it is at room temperature before removing the comb.
Remove the comb at a slight angle and 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 puncture the bottom with the pipet tip.
Samples not running If the comb is warped, replace.
along a straight path
If the running tray is warped, replace.
(Cool agarose to 50 °C to prevent the tray from warping.)
Circulate buffer if it becomes depleted by stopping
the run and pipetting the buffer from one chamber to
the other.
Double-banded pattern Make sure the comb remains vertical after the gel is cast
so that the well shape is not distorted.
Decrease the buffer level to 1 mm above the top of the
gel in order to reduce the temperature gradient in the gel.
Poor band resolution Add Ficoll ™ , glycerol, or sucrose to the sample loading
buffer to ensure that the sample sinks to the bottom of
the well. (Ficoll is the recommended agent.)
Make sure the sample is completely dissolved.
Reduce the sample concentration.
Reduce the sample volume.
Reduce voltage to 5 V/cm.
Be sure the well floor is at least 1 mm thick to prevent
samples from leaking through the 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.
Foam pads peel off Do not press the running tray into place. Install as
described on page 4.