1. What is the stability of the fluorescent products compatible to B-BOX™, and will they degrade?
Each product is uniquely different. If the product is kept frozen and stored away from light, in accordance to product specifications it will last >12 months.
2. After running the gel with the DNA sample and the fluorescent loading dye, would the fluorescent loading dye affect the sample making it inoperable in subsequent experiments?
Using the product does not affect subsequent operations. This is because the fluorescent dye can easily be removed by regular alcohol precipitation or gel elution kit.
3. Will the fluorescent loading dye affect cloning efficiency?
When used under normal blue-ray observation, and with plastic cutting, cloning efficiency is even better than that of EtBr and UV light. The experiments also indicate using the fluorescent dye coupled with blue light, the cloning efficiency is increased by about a 100 times.
4. Is it possible to use FluoroStain™ DNA Fluorescent instead of ethidium bromide (EtBr) in staining of DGGE gels which is composed of poly acrylamide?
In the case of DGGE staining with FluoroStain™ DNA Fluorescent Staining Dye (DS1000
), we can give some suggestions as stated below:
A. Similar to ethidium bromide, DS1000
could be used for staining DNA in polyacrylamide gels.
B. However, it is noted that the DS1000
is designed to stain dsDNA and thus is not appropriate for staining in DGGE due to denaturing of dsDNA.
C. For detection of ssDNA or RNA in DGGE, we strongly recommend the use of FluoroVue™ Nucleic Acid Gel Stain (NS1000) which can be used to stain both double-strands and single-strand nucleic acids.
D. Furthermore, it’s suggested to stain the gel after electrophoresis (post-staining) instead of pre-staining or in-gel staining, since the fluorescent dye might be affected during acrylamide polymerization.
E. The optimal excitation wavelength of DS1000
is around 480~490 nm (blue light), and therefore the illuminator with blue light is preferred. Of course, UV could be used also, but the signals detected might be weaker.
5. Can the SDS-PAGE gel stained with protein fluorescent staining dye (PS1000, PS2000) be transferred to perform Western Blot? Will it affect the antibody binding when Western Blot is performed?
After using protein fluorescent staining dye (PS1000
) to stain the gel, it is NOT recommended to perform Western Blot. The proteins in the SDS PAGE gel are fixed during staining with acidic solution, and thus preventing the transfer of proteins to NC or PVDF membrane.
6. In using FluoroStain™ DNA staining dye (DS1000), which method is recommended as the gel soaking and in-gel staining methods (pre-casted gel with fluorescent dye) are taken into account?
FluoroStain™ DNA staining dye (DS1000
) should be used by gel soaking (stain the gel after electrophoresis). For in-gel stain, FluoroVue™ Nucleic Acid Gel Stain (NS1000
) is suggested.
7. As the fluorescent DNA loading dye DL5000 is used, is the migration of DNA affected by the fluorescent dye?
Yes, it might be affected due to variation of DNA amount mixed with the fluorescent loading dye. The fluorescence composition in a fluorescent loading dye is fixed, so for different amounts of DNA different degrees of binding to fluorescent dyes are expected. Variety in DNA/fluorescence ratio might lead to variation in DNA migration; the higher the fluorescence ratio is, the slower the DNA fragments migrate. In general, a DNA amount of ≥31.25 ng with 1 μL DL5000
loading dye will be an acceptable range. If the DNA concentration is lower than 31.25 ng, the molecular weight determination may be slightly distorted. For analyzing DNA sample of unknown concentration, it is recommended to use the FluroStain™ DNA staining dye with post-staining method or to use FluroVue™ nucleic acid staining dye with in-gel staining method.
8. After post staining using the DNA fluorescent staining dye, why the DNA bands are sometimes blurred up and the fluorescent signal is not enough?
There are three possible reasons:
A. The fluorescent dye is very sensitive, so a small amount of smear due to DNA quality may blur the band.
B. The agarose is impure, or incompletely dissolved. Tailing effect may also cause the fluorescent signal to weaken.
C. There may not be enough dye or the staining time is insufficient. After each staining, gels will absorb a large amount of fluorescent dye, thus causing insufficient staining for the next time if the same staining bath is repeatedly used. Furthermore, the time needed for staining can be elongated based on the percentage and the thickness of the agarose gel; harder and thicker gels require a longer staining period.
9. Do you recommend using DNA staining dye diluted in loading buffer?
Ans.: We do not recommend this because the wrong concentration after dilution may cause inconsistent results.
10. Can we use white light boxes plus blue cellophane to observe fluorescent signals?
Not recommended because of limited intensity of blue light provided by a white light system.
11. How do we take pictures when we use the SMOBIO fluorescent dye?
Most of SMOBIO’s fluorescent dye can be viewed using blue light (B-BOX™) or UV lamp light source. If you need to take a picture, a regular UV image system or a smart phone can be used to take the picture from B-BOX™. However, if reflection in the picture is a problem, it is recommended to close the nearby light source or take the picture in a dark room.
12. Since SMOBIO's fluorescent dye products are all safer (non-mutagenic) to use, are there relevant documents that support their safety?
Our DNA staining products are congruent to Cyanine dyes. The scientific community generally believe Cyanine dyes are safer than EtBr. Currently, our fluorescent dyes either for nucleic acids or for proteins are proofed for their safety (non-mutagenicity) using Ames test (Figures below). However, it must be noted that since solvent may penetrate the skin, it is recommended that users wear gloves when using the fluorescent dyes.
13. When doing a follow up analysis using an SDS PAGE stained with fluorescent protein staining dye (PS1000), will there be any effect on mass analysis?
There will be no effect on mass analysis when following up with an SDS PAGE analysis using protein fluorescent staining dye (PS1000
14. Can you use DL5000, DS1000, NS100 dyes to detect nucleic acids under conventional UV light?
Yes, it is possible to view the fluorescent signal under blue light and UV light.
15. Does the quality of agarose gel matter when using FluoroVue™ Nucleic Acid Gel Stain (NS1000) or FluroStain™ DNA fluorescent staining dye (DS1000)?
Yes, insufficient quality of agarose gel may lead to the limited performance of the dye.
16. What is the generally recommended dilution of FluroStain™ DNA fluorescent staining dye (DS1000)?
A dilution of 10,000 times. For example, 10 μL staining dye is added to 100 mL of water, TAE, or TBE. Next soak the agar gel in the solution. Soaking time depends on the percentage and thickness of agarose gel.
17. Does using DS1000 for staining the DNA cause bias in DNA molecular weight determination?
No, when the gel is stained with DS1000
after electrophoresis, the DNA molecular weight interpretation is accurate.
18. How many ways can the SMOBIO fluorescent DNA staining dyes DS1000 or NS1000 be used?
For using the DS1000
, it is recommended to be used with post staining method. If DS1000
is used with in-gel staining or staining during electrophoresis, the DNA band may not be clear enough and DNA migration may be changed. For NS1000
, it can be used for all three methods.
19. Why does the gel stained with DS1000 or NS1000 show minimal background which is barely seen as compared with DNA/RNA signals that are strongly fluorescent after staining?
dyes manifests fluorescent signal when it properly binds with double stranded DNA. The NS1000
exhibits fluorescent signal when it binds with nucleic acid (dsDNA, ssDNA, RNA).
20. Can DS1000 dye be used to observe RNA?
The dye is designed specifically for double strand DNA staining not for RNA use. If there is the need to stain RNA, it is recommended to use FluoroVue™ nucleic acid stain, NS1000
, to stain RNA.
21. When using B-BOX™ with DS1000 dye, what is the improved cloning efficiency?
In most experiments, with proper use, you can increase your cloning efficiency to hundreds of colonies per late after transformation, as compared to traditional EtBr/UV light which allows single digit colonies only.
22. Before ligation, do we need to remove the staining dye?
DNA after general gel elution or alcohol precipitation can be directly used for ligation. It does not need any extra removal steps.
23. Why are the DNA signals not visible when using the DL5000, but the signal is strong with EtBr staining?
creates a signal that is bright enough. Probable causes for a weak signal are: it was not protected against light during electrophoresis and it was not viewed immediately after electrophoresis. Light will cause fluorescent dye degradation that results in weaker fluorescent signal. If the DL5000
is used with the correct procedure and you cannot see sufficient fluorescent signal, it may be due to the storage problem. The DL5000
needs to be protected from light and be stored in a 4 °C environment. It is not suitable for long-term storage in room temperature. Since the DNA staining dye is sensitive to light, so improper use, storage or transportation may diminish its signal.
24. Is PS1000 able to stain a 2D gel?
can stain a 2D gel, which is quite sensitive. PS1000
is capable of achieving detection levels parallel to silver staining. The sample gel can be used for further MASS analysis.
25. Can the DS1000, NS1000 or DL5000 be removed by general gel elution kit?
Yes. The qualities of different gel elution kit are similar for removing the staining dye. Therefore, there is no need to use high price elution kit. However, different elution kits may have different elution efficiency. After elution or purification there is no effect on subsequent experiments.
26. During the dilution of DS1000, it‘s occasionally seen that there are few red flocculent unable to be completely dissolved in the buffer. Is there any method to improve this?
To avoid the solid precipitation of DS1000
during -20℃ (4℃) storage, we recommend that after using vortex, make sure that solid particles are completed dissolved. In the event of incomplete dissolution, we recommend 100X dilution in water then 100X dilution in buffer.
27. DS1000 Retention period: Mentioned -20℃ and over 2 years. Is it possible to have a retention period of 30 months or 36 months? Have you tested this period?
In our tests, the DS1000
’s retention period could extend beyond 2 years if it is maintained at the recommended temperature of -20℃. However, we must recommend that the retention period should still be 2 years. Storage conditions should be in a dark room and temperature kept at -20℃. High temperature and light will result in fluorescent dye attenuation. We have tested our DS1000
at -20℃ for 30 months and it did perform with similar efficiency. Again, we do not recommend a retention period of more than 24 months at -20℃.
28. How many cycles of freezing and thawing can the DS1000 handle? If 5 μL is used at once, will it have a usage of 100 times?
Our tests have shown that 100 freezing and thawing cycles will be appropriate for the DS1000
. In DS1000
's usage information, it is indicated that the fluorescent dye should be protected from light and kept at low temperatures. After using DS1000
, it should immediately be kept between 4℃ to -20℃ as fluorescent dye decays at a faster rate at room temperature. Please remember that any unthawed solution will cause the fluorescent dye concentration to be less after each use therefore reducing its sensitivity performance.
29. Is there any information on the ionic strength, pH stability, and optimum pH for DS1000?
stock is diluted in DMSO and does not have any special pH range. It is not advisable to use DS1000
in water. Therefore, it should be used with 1x TAE, TBE, and TE buffer at pH8.0 to gain the desired results. The optimum pH value for DS1000
is 7.5 to 8.0. Less than 7.5 and more than 8.0 may result in reduced sensitivity.
30. Can the staining dye (DS1000, NS1000, or DL5000) be removed from DNA? How can we do it?
The staining dye can be removed from DNA with traditional ethanol precipitation, PCR clean up kit, or the gel extraction kit. The ethanol precipitation method can follow conventional molecular cloning or follow the listed protocol.
A. Measure the volume of the DNA sample.
B. Add 1/10 volume of 3M sodium acetate, pH 5.2,(final concentration of 0.3 M) - The pH value of 3M sodium acetate must be adjusted with acetate not with HCl.
C. Mix well.
D. Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition).
E. Mix well.
F. Place on ice or at -20 °C for >20 minutes.
G. Spin at maximum speed in a microfuge for 10-15 min.
H. Carefully decant supernatant.
I. Add 1 mL 70% ethanol. Rinse and spin briefly. Carefully decant supernatant.
J. Air dry or briefly vacuum dry pellet.
K. Re-suspend pellet in the appropriate volume of TE, Tris buffer or water.
31. Can the stained DNA be used for enzymatic reaction like ligation, enzyme cutting, PCR and so on? Do you have any recommended methods like pretreatment?
After removing the staining dye from DNA, the DNA can be used for ligation, restriction enzyme digestion or PCR reaction. It is imperative that the DNA is maintained in good quality for bioassay after removing the stained dye. We recommend removing the DS1000
with the gel extraction kit or PCR clean up kit because these methods are convenient and has high efficiency. The very low amount of DS1000
does not affect ligation, enzyme cutting or PCR. However, the threshold is related to the enzyme systems and is case by case. For best results, we recommend to remove the staining dye from DNA before proceeding to the next step of the experiment.
32. How do we dispose of the staining solution? Is the disposal method for EtBr solution acceptable for this product?
We recommend that disposal be made in accordance to the local law. The freshly prepared DS1000
solution can be filtered through activated charcoal before disposal. The charcoal can then be disposed of by incineration. One gram of activated charcoal easily absorbs the dye from 10 liters of freshly prepared working solution.
33. We have to tell the organization the information of the hazardous waste. Is the staining agent cyanine like dye?
Yes, the DS1000
is a kind of cyanine dye. The SYBR staining dyes, Cy3 and Cy5 are cyanine dyes too and have been used in biotechnology for a long time. The DS1000
is safer than EtBr based on the Ames test. Therefore most people believe that cyanine dye is safer than EtBr. For now there are no direct reports showing that the DS1000
is carcinogenic, but we recommend that DS1000
stains always be handled with the same care given to other nucleic acid stains, such as ethidium bromide.
34. What is the stability of the fluorescent DNA staining dye? Is it stable in hot condition and does it use in-gel or post staining?
The FluoroStain™ DNA staining dye can be stored at -20℃ for at least 24 months. If it needs to be used frequently, it can be stored at room temperature or 4℃. The DS1000
is recommended to be used with post stain method. For in-gel staining the best choice would be to use FluoroVue™ Nucleic Acid Gel Stain (NS1000
35. When the NS1000 or EtBr is used for in-gel staining, why do some of the lowest bands become weaker in a longer agarose gel
This is because EtBr or the dye component of NS1000
keep moving toward the cathode in a direction opposite to the DNA migration. Therefore after electrophoresis the concentration of NS1000
or EtBr is very low near the anode end (the bottom) of the agarose gel, and some bands close to the bottom will be very weak in signal or even undetectable. It is recommend to reduce the electrophoresis time or use post staining method to improve the intensity of small fragments of DNA.