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DNA ladder not showing up on agarose gel?
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Posted by
Michael Geissler
DNA ladder not showing up on agarose gel?
Pushkal Sinduvadi Ramesh, I used SYBR Safe. I do not think it is past its date and it has not been exposed to light. I did everything as dark as I could which is why I am a little confused.
Pushkal Sinduvadi Ramesh, I used SYBR Safe. I do not think it is past its date and it has not been exposed to light. I did everything as dark as I could which is why I am a little confused.
Hello Matthew Czmer How much Ethidium bromide did you add? I would suggest you add at least 5uL of 10mg/mL stock EtBr for a gel cast from 50mL agarose. By looking at your gel image I feel either you have used a very low concentration of EtBr or your stock EtBr was exposed to light and lost its ability. Hope this helps.
Hello Matthew Czmer How much Ethidium bromide did you add? I would suggest you add at least 5uL of 10mg/mL stock EtBr for a gel cast from 50mL agarose. By looking at your gel image I feel either you have used a very low concentration of EtBr or your stock EtBr was exposed to light and lost its ability. Hope this helps.
When I used Sybr Safe, I used to use it at 1:10000X dilution (10ul for 100ml Agarose). In any case, the bands for Sybr Safe were always very weak compared to EtBr in a UV transilluminator (which I think you might be using?), Sybr Safe is not really excited by UV like EtBr is. You should use a blue light transilluminator for Sybr Safe.
Also, these intercalating dyes are less sensitive to single-stranded NA species, so you might have to use even more dye. I also never wait for Agarose to cool down before adding EtBr/Sybr, they are quite heat tolerant and work perfectly fine even if boiled together with the Agarose (don't do this because of spill risks).
Honestly, I feel Sybr Safe is very much overrated - EtBr is in fact less toxic than Sybr Safe and also works under blue light transillumination pretty well. It's not the EtBr that hurts your DNA when gel-extracting, it's the UV.
When I used Sybr Safe, I used to use it at 1:10000X dilution (10ul for 100ml Agarose). In any case, the bands for Sybr Safe were always very weak compared to EtBr in a UV transilluminator (which I think you might be using?), Sybr Safe is not really excited by UV like EtBr is. You should use a blue light transilluminator for Sybr Safe.
Also, these intercalating dyes are less sensitive to single-stranded NA species, so you might have to use even more dye. I also never wait for Agarose to cool down before adding EtBr/Sybr, they are quite heat tolerant and work perfectly fine even if boiled together with the Agarose (don't do this because of spill risks).
Honestly, I feel Sybr Safe is very much overrated - EtBr is in fact less toxic than Sybr Safe and also works under blue light transillumination pretty well. It's not the EtBr that hurts your DNA when gel-extracting, it's the UV.
Hello, first clean the picture area in your imaging system and try a blank picture for 4s. Second may than help increasing dye concentration or try more RNA selective dye...
Hello, first clean the picture area in your imaging system and try a blank picture for 4s. Second may than help increasing dye concentration or try more RNA selective dye...
Pushkal Sinduvadi Ramesh, I used SYBR Safe. I do not think it is past its date and it has not been exposed to light. I did everything as dark as I could which is why I am a little confused.
Pushkal Sinduvadi Ramesh, I used SYBR Safe. I do not think it is past its date and it has not been exposed to light. I did everything as dark as I could which is why I am a little confused.
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Hello Matthew Czmer
How much Ethidium bromide did you add? I would suggest you add at least 5uL of 10mg/mL stock EtBr for a gel cast from 50mL agarose. By looking at your gel image I feel either you have used a very low concentration of EtBr or your stock EtBr was exposed to light and lost its ability.
Hope this helps.
Best!
Hello Matthew Czmer
How much Ethidium bromide did you add? I would suggest you add at least 5uL of 10mg/mL stock EtBr for a gel cast from 50mL agarose. By looking at your gel image I feel either you have used a very low concentration of EtBr or your stock EtBr was exposed to light and lost its ability.
Hope this helps.
Best!
More
VOTE
When I used Sybr Safe, I used to use it at 1:10000X dilution (10ul for 100ml Agarose). In any case, the bands for Sybr Safe were always very weak compared to EtBr in a UV transilluminator (which I think you might be using?), Sybr Safe is not really excited by UV like EtBr is. You should use a blue light transilluminator for Sybr Safe.
Also, these intercalating dyes are less sensitive to single-stranded NA species, so you might have to use even more dye. I also never wait for Agarose to cool down before adding EtBr/Sybr, they are quite heat tolerant and work perfectly fine even if boiled together with the Agarose (don't do this because of spill risks).
Honestly, I feel Sybr Safe is very much overrated - EtBr is in fact less toxic than Sybr Safe and also works under blue light transillumination pretty well. It's not the EtBr that hurts your DNA when gel-extracting, it's the UV.
When I used Sybr Safe, I used to use it at 1:10000X dilution (10ul for 100ml Agarose). In any case, the bands for Sybr Safe were always very weak compared to EtBr in a UV transilluminator (which I think you might be using?), Sybr Safe is not really excited by UV like EtBr is. You should use a blue light transilluminator for Sybr Safe.
Also, these intercalating dyes are less sensitive to single-stranded NA species, so you might have to use even more dye. I also never wait for Agarose to cool down before adding EtBr/Sybr, they are quite heat tolerant and work perfectly fine even if boiled together with the Agarose (don't do this because of spill risks).
Honestly, I feel Sybr Safe is very much overrated - EtBr is in fact less toxic than Sybr Safe and also works under blue light transillumination pretty well. It's not the EtBr that hurts your DNA when gel-extracting, it's the UV.
More
VOTE
Hello,
first clean the picture area in your imaging system and try a blank picture for 4s. Second may than help increasing dye concentration or try more RNA selective dye...
Hello,
first clean the picture area in your imaging system and try a blank picture for 4s. Second may than help increasing dye concentration or try more RNA selective dye...
More
VOTE