Dim Bands on Bottom Half of PCR Gel Using SYBR Safe?

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Subham Basak

Hi Jared Baas

The intercalating agents are positively charged and thus in the electrophoresis runs opposite to the DNA, finally giving you dimmer bands at the lower half of the gel.

I'm not sure if you are adding SYBR Safe to the gel or to your samples, but adding the visualizing agent to your samples (mixed with the tracking dye) can give better results.

Let me know if it helps,

Subham.

Sönke Weinert

Hi Jared Baas ,

I agree with Subham Basak . Another method to overcome the problem would be to stain the gel after the run. That will take (at least with EtBr) additional 10-15 minutes, but your gel would be stained uniform in the end.

Best wishes

Soenke

Nikolay Klyashtornyy

There are a few potential reasons why you may be seeing dim bands on the bottom half of your PCR gel when using SYBR Safe dye:

Uneven Gel Loading: If the gel was loaded unevenly, with more samples on the top row than on the bottom row, this could result in the bottom bands appearing fainter. Make sure to load the same amount of each sample in both rows.

Incomplete Gel Electrophoresis: If the gel is not run long enough, it is possible that the DNA fragments in the bottom row may not have migrated far enough down the gel to be visible. Make sure to run the gel for a sufficient amount of time, according to the size of your DNA fragments.

SYBR Safe Concentration: The concentration of SYBR Safe dye used can also affect the visibility of DNA fragments. If the concentration is too low, the bottom bands may not be as visible. Try using a higher concentration of SYBR Safe dye to see if this improves the visibility of your bands.

Gel Excision: If you are cutting out the bands from the gel, it is possible that the excision of the bands from the bottom row is not as efficient as from the top row. This could result in less DNA being extracted from the gel, and therefore less visible bands. Make sure to excise the bands carefully and completely from both rows.

Gel Drying: SYBR Safe dye can cause gels to dry out more quickly than other stains. If the gel dries out, this could result in the bottom bands appearing fainter. Make sure to keep the gel moist throughout the staining and visualization process.

By addressing these potential issues, you may be able to improve the visibility of the bands in the bottom half of your PCR gel.

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