DNA gel extraction / PCR product purification Product size < 15Kb

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. The resulting amplicons are generally detected by gel electrophoresis and for some further applications like cloning, sequencing, amplicon product needs to be recovered from the gel and subsequently purified. However, non-specific product amplification and primer-dimer formation during set-up make gel extraction difficult. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

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2 years ago

2 years ago by Stefan Fuhrmann Germany

Help with significant loss of DNA after extraction from gel

Hello everyone. I am in need of some help. After isolating DNA and amplifying it I ran it through an agarose gel to check it. When I tried to extract it from the gel the amount of DNA I got was too low to use. For instance, I started with around 500 ng/μl but managed to extract only concentrations as low as 10 ng/μl. What might be causing this significant loss? Also, I am wondering if purifying the pcr product with PEG mix is sufficient.

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Found 10 matching solutions for this experiment

Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Protocol tips
Remove the flow-through
by aspiration. Avoid
contamination of the
collection tube rim.
Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Protocol tips
Remove the flow-through
by aspiration. Avoid
contamination of the
collection tube rim.
Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Protocol tips
Remove the flow-through
by aspiration. Avoid
contamination of the
collection tube rim.
Purelink PCR Purification Kit

Thermo Fisher Scientific

Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Protocol tips
Remove the flow-through
by aspiration. Avoid
contamination of the
collection tube rim.
Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Protocol tips
Remove the flow-through
by aspiration. Avoid
contamination of the
collection tube rim.
Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
Upstream tips
The amount of agarose
excised from the gel should
be as small as possible.
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