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Q1 – Why is Midori Green safer than ethidium bromide or Sybr green?
There are three reasons:
1) Midori Green binds less aggressively to DNA molecules, so fewer mutations occur during synthesis.
2) Midori Green does not penetrate the skin or plasma membranes, so uptake into living cells is quite minimal.
3) Midori Green has a very short half-life, so it breaks down in the environment quickly.
The extensive testing on Midori Green bears this out.
Q2 – How can Midori Green be disposed of after use?
Midori Green does not create any toxic waste. It can therefore be disposed of according to laboratory regulations for non-toxic waste.
Q3 – Can I use my UV transilluminator or do I need to buy an LED table?
Yes and no. Midori Green Advance and Midori Green Direct do not require LED illuminators—as long as your UV table’s emission is 270 nm or higher results will be satisfactory. For best results we do recommend a blue LED or blue/green LED illumination source.
Midori Green Xtra is not compatible with UV illumination, but both blue LED and blue/green LED light sources will work very well. Please check our extensive range of FastGene Blue/Green LED illuminators and gel imagers.
Q4 – The bands on my gel look wavy when using Midori Green Direct. Can you explain that to me?
Midori Green Direct is a big molecule so that it won’t penetrate skin or cross plasma membranes. Because of its size, it is possible for this molecule to get stuck in the gel matrix. The “wavy bands” problem is caused mainly by excessive Midori Green Direct added to the sample, so we suggest using a 1:10 ratio of stain–and no more than 0.5 μL per sample, even if it’s less than 1:10!–to help ameliorate this effect.
Q5 – Is it possible to use Midori Green on acrylamide gels?
Yes. All three varieties of Midori Green can be used to stain nucleic acids on acrylamide gels.
Q6 – Can Midori Green be used with TAE and TBE buffers?
Yes. Both TAE and TBE buffer are compatible with Midori Green in agarose gels. Midori Green has good stability in these buffered solutions and will continue to provide a strong signal as long as the gels are maintained in light-proof storage containment.
Q7 – Is it possible to stain DNA with Midori Green in agarose gels as with EtBr (ethidium bromide)?
Yes. Midori Green can be used in the gel just like EtBr. However, post-staining should give a better sensitivity than precast gels, eliminating any possibility for the dye to interfere with the migration and separation of nucleic acid bands.
Q8 – Can Midori Green be used for post-staining?
Q9 – How should Midori Green Advance be diluted when used for post-staining?
Add 5-15 µL of Midori Green Advance to 100 mL of buffer solution. Adjust optimal concentration and staining duration (from 5 to 60 minutes usually) according to your experiment.
Q10 – Does Midori Green migrate in the gel?
Yes, being positively charged, Midori Green will run in the opposite direction as compared with DNA and RNA bands. This can result in a light/dark background transition. We recommend using optimal electrophoresis time and % of agarose for running gels. For longer products/run time we recommend post-staining.
Q11 – Can Midori Green be diluted?
Yes. Midori Green solution can be diluted in ultrapure water.
Q12 – Can we use Midori Green for RNA as well as DNA?
Yes. All three varieties of Midori Green produce very strong signals for RNA, dsDNA, and ssDNA.
Q13 – Which filters (wavelength) can be used for Midori Green?
Filters designed for green dyes (like a GFP/SYBR green filter) are the most sensitive. For Midori Green Advance and Midori Green Direct, an EtBr filter or just UV-light without any filter are also acceptable.
Q14 – Is it possible to add Midori Green to agarose dissolved in TAE buffer during the heating process?
No. Adding Midori Green to hot agarose solution is not recommended. We advise cooling the agarose solution to approximately 60°C, adding Midori Green, and then shaking carefully and mixing thoroughly.
Q15 – Does Midori Green detect low DNA concentrations?
Yes. Midori Green performs well with low DNA concentrations, as well as with small DNA fragments (≥100 bases).
Q16 – Does Midori Green interfere with downstream applications?
It may, depending upon the application. To avoid Midori Green in downstream processing steps you may first purify the DNA with a purification kit.
Q17 – Can Midori Green be used for pulse field gel electrophoresis?
No. This application is not recommended.
Q18 – Is it possible to use Midori Green with Southern blot experiments?
Yes. Midori Green is the perfect choice of DNA staining dye before blotting for Southern experiments. It will not interfere with transfer to a blot or hybridization with a probe.
Q19 – Does Midori Green interfere with recovery of DNA from the stained gel once it is run?
No. Midori Green does not typically co-purify with DNA or RNA.
Q20 – Where should Midori Green be stored?
For maximum performance and stability, Midori Green should be stored in the dark (Midori Green is provided in brown vials).
Q21 – Does Midori Green penetrate latex gloves?
No. Midori Green molecules are too large to penetrate latex gloves.
Q22 – Why doesn’t the sensitivity of Midori Green seem as high as that of ethidium bromide (EtBr)?
Emitted Light Filtering – We find that many researchers have optimized imaging/filtering conditions for EtBr, which is a red dye. Please pay close attention to the filters; though EtBr filters will work if there are no other options, they may absorb a lot of emitted shorter wavelength light and therefore lower the observed sensitivity. A GFP/Sybr Green filter is preferred for Midori Green dyes.
Excitation Light Source – Though transilluminators that emit shorter wavelengths (254 nm) are very good for EtBr, they are not good for Midori Green dyes. We recommend UV tables with emissions at 306 nm or higher–the longer the better! For optimal results, use blue LED and blue/green LED light sources for Midori Green Advance and Midori Green Direct. Midori Green Xtra is not compatible with UV; it should be used with blue LED or blue/green LED illumination only.
Natural Light Exposure – Midori Green is sensitive to photo-bleaching effects from long exposure to direct (or even indirect) sunlight. We recommend that gels be stored in a dark box or covered by aluminum foil if you do not plan to image them immediately.
Q23 – I see that when using Midori Green Advance, the bottom of the gel looks darker after electrophoresis. Why is this?
Midori Green is positively charged, so free dye in the gel matrix will migrate toward the top of the gel and create a darker front. This effect is only seen when Midori Green Advance or Midori Green Xtra are added to molten agarose. To give a more consistent background, you can post-stain or instead use Midori Green Direct (which gets added directly to the sample).
Q24 – The background fluorescence of Midori Green Advance in the gel is too high and makes it difficult to see the fainter DNA bands. Is there a recommendation to improve signal-to-noise for these faint bands?
Yes. If you are still seeing high background using 4 µL of Midori Green Advance in 100 mL of agarose gel, then go down to 2 µL. This should only lower background signal; faint bands will still be saturated with dye. This is especially effective with our Blue/Green LED technology, which gives a much brighter signal for all green and red dyes.
Q25 – Will Midori Green cause the migration rate of my DNA bands to change?
No—with one exception. Midori Green Advance and Midori Green Xtra will not alter the migration rate of DNA bands. On the other hand, if too much Midori Green Direct is added to a sample, it may cause bands to migrate differently. As a result, though we generally recommend using a 1:10 dilution with the sample, the amount should never exceed 0.5 µL of Midori Green Direct per sample. By following this rule of thumb your bands should not experience any unwanted shifts.