[DL5000] FluoroDye™ DNA Fluorescent Loading Dye (Green, 6X), 1 ml

Using FluoroDye™ DNA Fluorescent Loading Dye (DL5000) 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.

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 fluorescent staining dye 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 fluorescent staining dye from DNA before proceeding to the next step of the experiment. 

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 FluoroStain™ DNA staining dye with post-staining method or to use FluoroVue™ nucleic acid staining dye with in-gel staining method.