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Q1 – Why is Midori Green safer than ethidium bromide or Sybr green?
There are three reason for this:
1) Midori Green binds less aggressively to the DNA molecules, so fewer mutations occur during synthesis.
2) Midori Green does not penetrate the skin or the plasma membrane, 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. Therefore, it can be disposed of according to laboratory regulations for non-toxic waste.
Q3 – Can I use my UV transilluminator or do I need to buy the LED tables?
Yes. Midori Green dyes do not require an LED illuminator. If you would like to use a blue LED illuminator, we recommend our FastGene Blue LED systems or our FastGene GelPic LED Box.
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 into skin or cross the plasma membrane of cells. Because of the size, it is possible for this molecule to get stuck in the gel matrix. The wavy bands problem is caused mainly by quantity of Midori Green Direct added to the sample, so we suggest using a 1:10 ratio and no more than 0.5 μl per sample to help ameliorate this effect.
Q5 – Is it possible to use Midori Green on acrylamide gels?
Yes. Midori Green Advance and Direct can be used to stain nucleic acids on acrylamide gels.
Q6 – Can Midori Green be used with TAE and TBE buffers?
Yes, when running agarose gel, the electrophoresis buffer may be TAE or TBE buffer.
Q7 – Is it possible to stain DNA with Midori Green in agarose gels as with EtBr (ethidium bromide)?
Yes, it can be used with the DNA in the gel 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 thus the separation of the nucleic acid bands.
Q8 – Can Midori Green be used for post-staining?
Q9 – How should Midori Green be diluted when used for post-staining?
Add 5-15 µl of Midori Green in 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, therefore it is recommended to use optimal electrophoresis time and % of agarose for running gels. For longer products/running-time we recommend to use post-staining.
Q11 – Can Midori Green be diluted?
Midori Green solution can be diluted in the ultrapure water.
Q12 – Can we use Midori Green for RNA as well as DNA?
Midori Green can be used for RNA as well as dsDNA and ssDNA.
Q13 – Which filters (wavelength) can be used for Midori Green?
The filters designed for green dyes (like GFP/SYBR green filter) are the most sensitive. However, EtBr filter and simply UV-light without any filter can be used as well.
Q14 – Is it possible to add Midori Green when agarose is dissolved in TAE buffer with on the hot plate and magnetic stirrer during the heating process?
It is not recommended to add Midori Green to hot agarose solution. We recommend after making agarose solution, cool it down to approximately 60°C, then add Midori Green, shake carefully and mix thoroughly.
Q15 – Does Midori Green perform with low concentration of DNA?
Yes, Midori Green can perform with low concentration of DNA or small DNA fragments (100 bases)
Q16 – Does Midori Green interfere with downstream applications?
Avoid leaving Midori Green in downstream solution. You may purify the DNA with a purification kit.
Q17 – Can Midori Green be used for pulse field gel electrophoresis?
It is not recommended to use Midori Green for pulse field gel electrophoresis.
Q18 – Is it possible to use Midori Green with Southern blot experiments?
Yes. Midori Green is the perfect choice as a DNA staining dye before blotting for a Southern experiments. It will not interfere with transfer to a blot or hybridization with a probe.
Q19 – Does the stain interfere with recovery of DNA from the stained gel once it is run?
No. Midori Green should not cause any problems for further experiments with that DNA.
Q20 – Where should Midori Green be stored?
For the maximum performance and stability Midori Green should be stored in the dark (Midori Green is provided in brown vials).
Q21 – Does Midori Green penetrate the latex gloves?
Midori Green should be impenetrable to latex gloves.
Q22 – Why doesn’t the sensitivity of Midori Green seem as high as ethidium bromide (EtBr)?
Emitted Light Filtering – We find that many researchers have optimized imaging/filtering conditions for EtBr, which is a red dye and not a green one , like Midori Green. Please pay close attention to the filters which may absorb a lot of emitted shorter wave-length light and therefore lower the observed sensitivity. A 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 306 and higher wavelength (the longer, the better). Blue LED tables will work very well for Midori Green Direct only.
Natural Light Exposure – Midori Green is sensitive to photo-bleaching effects by long exposures with 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 immediately.
Q23 – I see that when using Midori Green Advanced 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 creating a darker front. This effect is only seen when Midori Green is added to molten agarose. To give a more consistent background, you can stain with Midori Green post run or use Midori Green Direct.
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 reccomendation 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 as faint bands will still be saturated with dye. This will especially the case if you use 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?
Midori Green Advance will not alter the migration rate of DNA bands. On the other hand, if too much Midori Green Direct (the second type of Midori Green) is added to a sample, then this can cause bands to migrate differently. That is why we recommend using the lesser of either: i) 0.5ul of Midori Green Direct per sample or i) diluting Midori Green Direct to a 1:10 concentration with the sample. By following this rule of thumb your bands should not experience any unwanted band shifts.