Quantitation Of Isolated Dna; Troubleshooting - Thermo Scientific invitrogen E-Gel G8121ST User Manual

Quantitation of isolated DNA

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Troubleshooting

For common E-Gel troubleshooting guidelines refer to troubleshooting guide (see page 36).
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Observation
Poor resolution or
smearing of bands
Low yield
Target DNA band cannot
be seen
DNA band passed the
recovery gel
DNA migration exhibits
smiley effect
E-Gel Power Snap Electrophoresis System User Guide
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Recovered DNA can be assessed using the Qubit
gel electrophoresis.
qPCR is recommended for accurate quantitation of next generation sequencing libraries
recovered from E-Gel SizeSelect
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Recovered samples are not compatible with 280 nm measurements without first
performing buffer exchange.
Cause
Sample is overloaded
High salt concentration
Total sample volume is too low
or too high
Loading wells were not pre-
filled with deionized water prior
to loading the sample
Samples were not prepared
properly
Incorrect loading volume
chosen
Recovery wells were not filled
with water prior to elution
Target DNA passed the
recovery gel
DNA amount is too high
High ambient light or low
sample amount
Selected protocol time was too
long
Extended gel run time or aged
gels used or incorrect loading
conditions
fluorometer (Cat. No. Q32868), or by
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II gels.
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Recommended action
Do not exceed 500 ng of total DNA per one sample
lane or 500 ng DNA per one band. Do not exceed
1 µg for sheared DNA.
Dilute your samples 2- to 30-fold depending on the
E-Gel type.
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Use recommended sample volume of 25 μL per
lane.
Fill all gel wells with 50 μL of deionized water prior
to loading any sample or a ladder.
Prepare up to 25 μL of sample in 1X concentration
of 10X Sample Loading Buffer.
Load up to 25 μL of prepared sample per well.
Once target fragment reaches reference line, pause
the run and fill all recover wells with deionized
water.
Carefully observe the DNA migration into the
recovery well. Minimize ambient light or perform
the workflow in dark room.
Collect DNA from the well in two or more fractions.
Be sure to load the recommended DNA amount.
Perform the workflow in dark room environment to
minimize ambient lights.
Choose the Reverse E-Gel program to run the band
backwards into the collection well.
Do not run gels longer than 30 minutes. Use fresh
gel. Follow sample loading recommendations.
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