SafeGel green stain for DNA electrophoresis

Question 1

Can I use the same filters and camera settings for gels with SafeGel as for EtBr?

Answer

Yes, you can use the same filters and camera settings of your imaging system for SafeGel as for gels with EtBr. Please note the emission spectra of SafeGel for specific wavelengths. SafeGel and EtBr have virtually the same emmision spectrum, so you can replace EtBr directly with SafeGel without having to change your existing imaging system.

Question 2

How sensitive is SafeGel?

Answer

SafeGel is far more sensitive than EB without requiring a destaining step.

Question 3

How stable is SafeGel?

Answer

SafeGel is available as a 10000X aqueous solution. It is stable at room temperature (+15 °C to +30 °C) for long-term storage. It can be microwaved but this is not recommended.

Question 4

How to dispose SafeGel?

Answer

Since SafeGel is non-toxic, non-mutagenic and non-hazardous when used at the recommended working concentration, it can be easily disposed of with normal waste. Separate handling of the waste as with EtBr is therefore not necessary. If necessary, obtain information from your local waste disposal company.

Question 5

How should SafeGel be stored?

Answer

SafeGel should be stored at room temperature (+15 °C to +30 °C) protected from light.

Question 6

How should SafeGel be used?

Answer

SafeGel is used for pre- and post-staining of agarose gels. We recommend using SafeGel in an in-slot application for best results.

Question 7

Can SafeGel also be used for polyacrylamide gels?

Answer

Yes, SafeGel can also be used to stain dsDNA, ssDNA or RNA in polyacrylamide gels in the post-staining protocol.

Question 8

Can SafeGel also be used for formaldehyde, DGGE, EMSA or PFGE gels?

Answer

Yes, SafeGel can be used for formaldehyde gels for staining RNA in the precast-staining protocol. For DGGE, EMSA and PFGE gels we recommend the post-staining protocol.

Question 9

Is an additional loading buffer needed when using SafeGel?

Answer

Yes, an additional loading buffer is needed. We routinely use 6X loading buffer containing 15% glycerol, 7.5% Ficoll 400, 0.05% Bromophenol Blue. In internal testing 6X loading buffer containing 0.1% Orange G produced good results in precast and post-stained gels. SDS in loading buffer may contribute to band smearing in precast SafeGel gels. If this occurs, we recommend using the post-staining protocol.

Question 10

Does SafeGel red stain need to be used in the dark?

Answer

No, you can use the dye in room light; however, we recommend storing the dye in the dark.

Question 11

How should I dilute SafeGel red stain for an in-slot application?

Answer

We recommend in-slot application with a 20X SafeGel red stain sample mix.

Question 12

What is the lower detection limit of SafeGel red stain?

Answer

Some users have reported being able to detect less than 0.1ng DNA. However, the limit of detection will depend on instrument capability and exposure settings.

Question 13

What is the binding mechanism of SafeGel red stain?

Answer

SafeGel red stain most likely binds by a combination of intercalation and electrostatic interaction.

Question 14

What is the chemical structure of SafeGel red stain?

Answer

The chemical structure of SafeGel red stain is proprietary.

Question 15

Does SafeGel red stain migrate during electrophoresis?

Answer

SafeGel red stain does not migrate through the gel as easily as EtBr. It is not necessary to add dye to the running buffer, and the gel will be stained more homogenously with SafeGel red stain than with EtBr.

Question 16

Can SafeGel red stain be removed from the DNA again?

Answer

Yes, SafeGel red stain may be removed from DNA by phenol/chloroform extraction and ethanol precipitation.

Question 17

Does SafeGel red stain interfere with further processing of DNA?

Answer

No, SafeGel red stain does not interfere with further processing of the DNA such as restriction enzyme, Southern blotting techniques and cloning.

Question 18

Is SafeGel red stain compatible with downstream DNA applications?

Answer

Yes, SafeGel red stain is compatible with downstream DANN applications such as cloning, ligation and sequencing. We recommend gel extraction kits, Exo-Sap protocol or phenol-chloroform extraction to remove the dye from DNA. Some users have reported performing PCR on DNA in the presence of SafeGel red stain with no purification step.

Question 19

Can SafeGel red stain be used in cesium chloride gradients?

Answer

Yes, customer have reported using SafeGel red stain in cesium gradients. To extract SafeGel red stain from DNA after cesium banding, we recommend adding SDS to a final concentration of 0.1% before butanol extraction. Alternatively, chloroform can be used instead of butanol for extraction.

Question 20

Is SafeGel red stain compatible with Southern or northern blotting?

Answer

Yes, SafeGel red stain can be used for blotting. We recommend using the post-staining protocol for blotting applications.

Question 21

Can I reuse SafeGel red stain pre-cast gel after electrophoresis?

Answer

Yes, it is possible, but we do not recommend reusing SafeGel red stain pre-cast gels as signal decreases with subsequent electrophoresis.

Question 22

SafeGel red stain is precipitated in the solution. Can SafeGel red stain still be used?

Answer

Dye precipitation may occur at lower temperatures (winter times), resulting in lower signal or the appearance of precipitate on the surface of the gel. If this occurs, heat the solution to 45-50°C for two minutes and vortex.

Question 23

I accidentally left my SafeGel red stain in the light. Will it still work?

Answer

While we recommend that you protect the dye from light during long term storage, we have had customers report using SafeGel red stain with success after accidentally leaving it in ambient light for more than one month.

Question 24

I have smeared DNA bands in the gel. What can I do?

Answer

1. Reduce the amount of DNA loaded by one-half to one-third. Blown out or smeared bands can be caused by overloading. This is frequently observed with DNA ladders. 2. Perform post-staining instead of pre-casting. 3. Pour a lower percentage agarose gel for better resolution of large fragments. 4. Change the running buffer. TBE buffer has a higher buffering capacity than TAE. 5. Loading buffers containing SDS may contribute to band smearing. If this occurs, use the post-staining protocol for applications requiring SDS-containing loading buffers.

Question 25

I have a discrepant DNA migration in pre-cast gel. What can I do?

Answer

SafeGel red stain is designed to be larger than other dyes to prevent it from entering cells, thus rendering the dye safer. The migration of DNA may be affected depending on the dye:DNA ratio. 1. Reduce the amount of DNA loaded by on-half to one-third. 2. Reduce the amount of dye used, i.e., use 0.5X in pre-cast gels. 3. Post-stain the gel in 3X SafeGel red stain to avoid any interference the dye may have on migration during electrophoresis.

Question 26

I observe a weak fluorescence, decreased dye performance over time, or film of dye remains on gel after post-staining. What can I do?

Answer

The dye may have precipitated out of solution. 1. Heat SafeGel red stain solution to 45-50°C for two minutes and vortex to redissolve. 2. Store dye at room temperature to avoid precipitation.

SafeGel green stain for DNA electrophoresis

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