We would like to run agarose gels without ethidium bromide to avoid the need of dealing with toxic waste and to reduce the use of dangerous chemicals in the lab. We talked to quite a few vendors about this and started trying things out.
We started with AMRESCO EZ-Vision dyes which are SYBR Green based and were not very happy with them. Staining was very weak and we could not reliably image the gels on our existing UV image acquisition system without a SYBR filter. We had no intention of buying a new image acquisition system just for this so we kept looking. 500uL $89.52 from VWR
We tried Promega Diamond Nucleic Acid dye was also pretty weak and it is not an in-gel stain, so requires additional time for post-staining. We decided to keep looking. 500ul $70.00 from Promega
Finally, we started having good results with Biotium GelRed. GelRed is very sensitive and looks orange/red on the UV transilluminator like EtBr. GelGreen was not quite as bright, so we focused on GelRed, but optimization was needed. We had two primary issues: 1) bands which are very close to each other appear like they are fused together and the molecular weight marker was just a smear, 2) bands sometimes ran at a different weight than expected.
Since one of our genotyping PCRs needs to distinguish between a 400 and 480bp band it was very important to get good separation. Biotium recommended to reduce the amount of dye in the gel or to try post-staining because both the separation issue and the run length issue could be due to the larger size of the Biotium dye which can impair DNA motility. We lowered the amount of in-gel stain from 10,000X to 40,000X and it still worked pretty well, but a range of 20-30,000X may work best (around 2ul for a 50mL gel). Another way to improve separation is to post-stain if necessary and post-staining is relatively quick (15-20 mins) and does not require destaining. In addition the post-staining dye can be reused saving some extra money. Because of the size of the dye, agarose concentration is also critical and concentrations above 1% are not recommended. 500ul $104.42 from VWR
All in all for regular genotyping PCR which is most of what we do the GelRed works very well and while it's somewhat more expensive than the other dyes, it is a substantial difference in sensitivity. In the end the decision comes to whether you want to have an EtBr free gel bench and how much money you are going to spend for the more expensive dye.
5g of EtBr ($64.20) will make 1L of 10,000X solution (final concentration 5mg/mL), while 1L of GelRed at 30,000X would cost $35,000. Yet, 1L of stock will make 100,000 100mL gels: if you run 10 100mL gels a week or around 500 gels a year, you'll spend $0.32 in EtBr or $417.68 in GelRed. I would be interested in finding out how much you spend yearly for EtBr disposal.
We started with AMRESCO EZ-Vision dyes which are SYBR Green based and were not very happy with them. Staining was very weak and we could not reliably image the gels on our existing UV image acquisition system without a SYBR filter. We had no intention of buying a new image acquisition system just for this so we kept looking. 500uL $89.52 from VWR
We tried Promega Diamond Nucleic Acid dye was also pretty weak and it is not an in-gel stain, so requires additional time for post-staining. We decided to keep looking. 500ul $70.00 from Promega
Finally, we started having good results with Biotium GelRed. GelRed is very sensitive and looks orange/red on the UV transilluminator like EtBr. GelGreen was not quite as bright, so we focused on GelRed, but optimization was needed. We had two primary issues: 1) bands which are very close to each other appear like they are fused together and the molecular weight marker was just a smear, 2) bands sometimes ran at a different weight than expected.
Since one of our genotyping PCRs needs to distinguish between a 400 and 480bp band it was very important to get good separation. Biotium recommended to reduce the amount of dye in the gel or to try post-staining because both the separation issue and the run length issue could be due to the larger size of the Biotium dye which can impair DNA motility. We lowered the amount of in-gel stain from 10,000X to 40,000X and it still worked pretty well, but a range of 20-30,000X may work best (around 2ul for a 50mL gel). Another way to improve separation is to post-stain if necessary and post-staining is relatively quick (15-20 mins) and does not require destaining. In addition the post-staining dye can be reused saving some extra money. Because of the size of the dye, agarose concentration is also critical and concentrations above 1% are not recommended. 500ul $104.42 from VWR
All in all for regular genotyping PCR which is most of what we do the GelRed works very well and while it's somewhat more expensive than the other dyes, it is a substantial difference in sensitivity. In the end the decision comes to whether you want to have an EtBr free gel bench and how much money you are going to spend for the more expensive dye.
5g of EtBr ($64.20) will make 1L of 10,000X solution (final concentration 5mg/mL), while 1L of GelRed at 30,000X would cost $35,000. Yet, 1L of stock will make 100,000 100mL gels: if you run 10 100mL gels a week or around 500 gels a year, you'll spend $0.32 in EtBr or $417.68 in GelRed. I would be interested in finding out how much you spend yearly for EtBr disposal.
So are you still using the GelRed and happy with the results? Thanks!
ReplyDeleteYes. We had to optimize some of our PCRs because it is so powerful you have to use very little DNA otherwise it's hard to see doublets, but it's been working for us for the past year and a half.
DeleteCool, thanks :)
ReplyDeleteWow! Thank you for sharing. ^^ I was just wondering, would it be necessary to load GelRed (as a loading buffer) with samples AND prestain the gel? Its okay if you're not really sure. :)
ReplyDeleteWe just add it in the gel like you would for EtBr and we use it really diluted, like 1:30,000 or 1:40,000. It's very sensitive. We don't add it to the samples. Too much dye makes the bands too thick and it's very hard to resolve doublets.
Delete• Thanks for sharing this valuable info!
ReplyDeleteAt Xitij Instruments we care about the users who care about finding the new things! And we care about finding new state of art instruments like UV Transilluminators and other lab equipments for our valued customers.