Electrophoresis with sybrgreen - (Jan/08/2008 )
Hello everyone,
in our lab we tried to used sybrgreen for real-time PCR but it didn't work. We still have some sybrgreen solution left and we want to use it for electrophoresis, instead of ethidium bromide. Does anyone have any experiences with sybrgreen in electrophoresis, advantages, disadvantages, maybe a recipe how to prepare a gel?
Thank you all for any information.
Ishia
Hello,
I used the "SYBR Safe DNA Gel Stain" from Invitrogen, and it worked quite good.
You can mix the SYBR with your agarose gel after you melted it, before to pour it in the gel box, like you do with EtBr.
Only think is that the SYBR Green do not shine in the same spectra as EtBr, and if you do not have a specific filter in your imager, the signal can appear a little bit fainter with SYBR Green as compared to EtBr.
Fabien
This dye should have higher affinity to DNA than EtBr. Can you just add to your DNA sample and run it on a gel without this dye? Has anyone test that?
Fabien, thank you for the information. We already have that filter. Could you just tell me what concentration of sybrgreen do you use?
thank you again
thank you again
Invitrogen recommends the identical amount for SYBR Safe as you would use with 10 mg/mL ethidium bromide. 5 uL of their 10000X solution in a 50 mL gel. We use 2.5 uL in 50 mL and that is fine.
I probably wouldn't do what you suggested Genehunter. It still has a negative charge and migrates towards the top of the gel.
We add sybrgreen directly into our samples without staining the gel as well, though with a rather unorthodox protocol. This works as long as enough sybrgreen is added to the samples. We dilute the 10.000x solution 1:200 in DMSO and add min. 1 µl per 5 µl PCR product. If too little is used and additionally the samples have different DNA concentrations they can migrate with different speed even if they have the same fragment size. When the gel runs for a long time (>1-2 h) the bands also start getting a little fuzzy.
We use this for checking PCR products for amplification etc. If we need the exact size of a fragment we use EtBr to be sure.
Instead of normal agarose gel loading dye (works too) we use the ALFexpressII loading dye with 99% formamide and 1% dextran blue to which we add a little extra BPB and XC so you can see the gelfront migration (dextran blue will stick to the wells). This is a dye for denaturated polyacrylamid gels; someone in our lab used it accidently for a while and it looked better than gels with the usual agarose gel dye. It lessens the different migration speed effect, but the bands will appear less bright under UV light.
So as a summary: it's possible, but you have to test it for a while and use enough sybrgreen to be sure of the results.