Using FluoroStain™ DNA Fluorescent Staining Dye (DS1000) does not affect subsequent operations. This is because the fluorescent dye can easily be removed by regular alcohol precipitation or gel elution kits. 

Using the fluorescent dye coupled with blue light, the cloning efficiency is increased by about 100 times compared to using EtBr and UV light.

FluoroStain™ DNA Fluorescent Staining Dye (DS1000) is 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.

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 staining 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 or NS1000 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. 

FluoroStain™ DNA Fluorescent 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

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. 

We do not recommend this because the wrong concentration after dilution may cause inconsistent results. 

Yes, insufficient quality of agarose gel may lead to the limited performance of the dye. 

A dilution of 10,000 times. For example, 10 μL staining dye is added to 100 mL of water, TE, TAE, or TBE. Next soak the agar gel in the solution. Soaking time depends on the percentage and thickness of agarose gel. 

No, when the gel is stained with FluoroStain™ DNA staining dye (DS1000) after electrophoresis, the DNA molecular weight interpretation is accurate. 

For using the FluoroStain™ DNA Fluorescent Staining Dye (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.

FluoroStain™ DNA Fluorescent Staining Dye (DS1000) manifests fluorescent signal when it properly binds with double stranded DNA

FluoroStain™ DNA Fluorescent Staining Dye (DS1000) 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. 

In our tests, the retention period of FluoroStain™ DNA Fluorescent Staining Dye (DS1000) 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℃. 

Our tests have shown that 100 freezing and thawing cycles will be appropriate for the FluoroStain™ DNA Fluorescent Staining Dye (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.

FluoroStain™ DNA Fluorescent Staining Dye (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. 

The staining dye can be removed from DNA with traditional ethanol precipitation, PCR clean up kits, or the gel extraction kits. The ethanol precipitation method can follow conventional molecular cloning or follow the listed protocol. Ethanol precipitation.

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.

After removing the fluorescent 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 fluorescent staining dye with the gel extraction kits or PCR clean up kits because these methods are convenient and high efficiency. The very low amount of DS1000 does not affect ligation, enzyme digestion or PCR. However, the threshold is related to the enzyme systems and it is on a case-by-case basis. For the best results, we recommend to remove the staining dye from DNA before proceeding to the next step of the experiment. 

We recommend that disposal be made in accordance to the local law. The freshly prepared FluoroStain™ DNA Fluorescent Staining Dye (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. 

FluoroStain™ DNA staining dye (DS1000) 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).