FAQ / Trouble Shooting
During Gel Preparation Step:
1)
Agarose solubility (seeing undissovled particle in melted
agarose):
It is critical that agarose is fully dissolved before casting. Undissolvedagarose results in high gel background or compromised gel resolutions after
electrophoresis.
To make sure agarose fully dissolved, first, completely soak
agarose powder in buffer prior to heating.
Set microwave on HIGH POWER, bring to boil and SWIRLin between
heating to fully dissolve clumping or particle adhering to side of flask.
For high concentration gel (≥2%),set microwave on MEDIUM POWER
and melt agarose slowly by repeating heating step (bring to boil, swirl, & reheat to boil) several
times. You may need to add additional hot DD water to compensate
evaporation during heating process.
The agarose is fully dissolved when you see CLEAR solution
after steps of heating and swirl.
Please view video at
http://www.hydragene.com/Protocol.html
for st
ep by step video
demonstration.
2)
Overspill of agarose during
heating:
Make sure total agarose solution occupies less than 50% of flask capacity. For high concentration gel (≥2%),it has tendency to easily over
boil. Stop when see signs of over boil and reheat in several steps (bring to boil, swirl,
& reheat to boil) in order to achieve fully dissolved CLEAR
solution.
During Electrophoresis Step:
1) Smeared DNA
bands:
Degraded DNA - Avoid nuclease contamination.
DNA overloading DNA -Decrease the amount of
DNA loading.
Aged running buffer – After many uses, aged buffer exhibit lowered ionic strength and
pH value with lowering buffering effect. Use fresh
buffer.
Improper electrophoresis conditions - Do not allow voltage to
exceed ~20 V/cm. Maintain a temperature <30¡ãC during electrophoresis. Make sure
electrophoresis buffer used has sufficient buffer capacity. For example, if 0.5X TAE/TBE
buffer used, suggest try 1xTAE/TBE.
Too much salt in DNA – Desalt DNA prior to
electrophoresis
DNA contamination with protein- remove protein prior to
electrophoresis
Denatured DNA - Do not heat prior to electrophoresis.
2) Faint or no bands on gel:
Insufficient quantity or concentration of DNA loaded - Increase amount of
DNA.
Degraded DNA - Avoid nuclease contamination.
DNA ranoutside of the gel – run gel for shorter time, use a lower voltage, or use a
higher percent gel.
Large size DNA – routine electrophoresis may not be
recommended. Suggest pulse field gel
electrophoresis.
Denatured DNA - Do not heat prior to electrophoresis.
Closely related size bands cannot be separated – increase electrophoresis time, and
/or use gel percentage suggestion below.
Separation of DNA in agarose
Agarose gel (%)
|
0.3
|
0.6
|
0.7
|
0.9
|
1.2
|
1.5
|
2.0
|
DNA size range (kb)
|
60-5
|
20-1
|
10-0.8
|
7-0.5
|
6-0.4
|
4-0.2
|
3-0.1
|
3) Abnormal DNA band migration or wavy
and smiley bands:
Buffer used for gel melting and gel running were prepared at
different times – use fresh buffer prepared at same time. For running
buffer, add enough buffer to cover gel 1-2mm above gel.
High voltage – First run at low voltage (3V/cm), once DNA
migrates outside of well, increase to normal voltage.
Loading and running too fast - Slowly load DNA and only run once sample lowered to bottom of
well.
Denatured DNA - Do not heat prior to electrophoresis.
HydraGene, Co. Ltd. is an
affiliated company of ACTGene, Inc.
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