INTRODUCTION
The kit contains a reagent for DNA labelling with a very high affinity fluorescent dye. This dye migrates almost completely linked to the DNA and is stable in the electric field applied during the electrophoretic separation. It does not diffuse in the gel or in the buffer but remains almost exclusively localised in the DNA bands. This allows a remarkable reduction of background and pollution, since MegaFluor will not contaminate the electrophoretic assembly and the electrophoretic buffer that can be poured directly in the sink without environmental pollution.

PURPOSE
Labelling the amplified DNA without the use of excess dye.
Avoiding the use of large amounts of highly harmful chemical compounds in the electrophoretic gels or buffers, like ethidium bromide Reduce the costs of decontamination and disposing of wastes.

TOXICITY
MegaFluor is a very big molecule, water soluble and not able to pass through cellular membranes. Studies performed on epithelial cells treated with MegaFluor, showed no traces of this molecule in cytoplasm or in the nucleus of these cells after incubation in medium with MegaFluor.

KIT CONTENT
A. MegaFluor - DNA Staining Solution 1.5ml (enough to run 1.500 DNA or RNA samples of 10ml)
B. Gel and Running Buffer 20x - 280 ml, enough to run at least 50 mini gels (25 ml gel volume)
C. Loading Buffer - 5ml (enough to run 1.500 DNA or RNA samples of 10ml)

METHOD
1. Open the microtubes add MegaFluor to each sample in the proportion of 1:10 and the Loading Buffer to each sample in the proportion of 2:10, mix and incubate for 5 minutes at 60°C (not shorter). Double the quantity to stain your DNA markers solution.
2. Prepare a 1x buffer by diluting the 20x gel buffer with deionized water and use it for the agarose gel preparation
3. When the agarose gel is well formed submerge it in the 1x buffer as a running buffer, which may be reused 3 times without any change in performances.
4. Fill the agarose gel wells with the labelled samples and perform your electrophoretic run as usual
5. Visualise and photograph the amplification products on an UV transilluminator as you are use to do with ethidium bromide.

STORAGE
+4°C

NOTES

- Using MegaFluor, the band due to PCR unincorporated primers can be more intense than usual or even split in two. This does not interfere with the separation and is due to proper additives that remove any excess dye and block it in the gel
- due to a lower background, photographing gel, exposure time can be increased compared to ethidium bromide labelled DNA
- Protocol Step 1. is very important for a correct labelling of nucleic acids: always incubate at 60°C for not less than 5 minutes.
- MegaFluor intercalates only double stranded nucleic acids: it is ideal to label either DNA or RNA in native gels (both agarose and acrylamide), but it is not recommended in denaturing conditions.