DNA che macchia il metodo basato su Formazan precipitazione indotta dall'esposizione alla luce blu

The DNA staining protocol has three general steps. The first is to put the gel in a solution of SYBR Green I and nitro blue tetrazolium, also known as NBT. The gel is then stirred for 25 minutes if it is a polyacrylamide gel and an hour if it is an agarose gel.

The last step is to expose the gel to a blue light source such as blue LEDs or sunlight. During this step some precipitate bands appears. These bands have a direct correlation with the DNA concentration and the gel then can be digitalized in an office scanner in order to quantify the DNA.

For the polyacrylamide gel, a non-denaturing polyacrylamide gel is prepared. The gel is then loaded with the samples and the electrophoresis process starts. SYBR Green I being diluted to 1.2X and the NBT 20 millimolar solutions are now prepared.

The gel is put into a plastic container and 16.75 mils of the diluted SYBR Green I and 325 mils of the NBT at 20 millimolar are added. Then the container is covered with Parafilm and aluminum foil and stirred at 60 rpm for 25 minutes. Finally the aluminum foil and a Parafilm is removed and the gel is exposed to sunlight or a blue light source.

For band quantification and calibration curves, the gel is put into an office scanner, then the image is analyzed using an image analysis software. Finally, a graft of normalized signal versus concentration is made. An agarose gel is made.

The wells are loaded with a plasmid that has ampicillin resistance and the gel is cut into two pieces, each of which is stained with a different stain. Then the plasmid band is extracted and transformed via chemical transformation into competent cells with media that contains ampicillin. A 1%agarose gel is prepared in TAE 1X buffer and loaded with four microliters of plasmid at around 100 nanograms per microliter.

The gel in the electrophoresis chamber is then run at 100 volts for one hour. Half the gel is stained with SYBR Green I and NBT method and the other half with SYBR Green I UV method. For agarose gel, the SYBR Green I NBT method is performed using SBYR Green I at a concentration of 1.2X and NBT at 2 millimolar.

The gel is put into a plastic container and the solution volume is scaled to cover the gel and the stirring time is extended to one hour. For agarose gels, the commonly used SYBR Green I method is performed by covering the gel with 1X SYBR Green I solution and stirring for an hour. Then the gel is exposed to a UV transilluminator for band visualization for two minutes.

Once the gel is stained, the plasmid band is cut with a scalpel and the plasmid extracted with a commercial extraction kit. The DNA is normalized to 9.1 nanograms per microliter for transforming chemical competent Escherichia coli XL 10 bacteria. Finally, the colonies are counted.

The colony-forming units are calculated. The result is grafted and the results obtained from SYBR Green I NBT and SYBR Green I UV compared. A purple formazan precipitate appears where the DNA is located.

In the experiment a DNA ladder was loaded to make a calibration curve with a detection limit of 192 picograms at 1, 500 base pairs. The use of agarose gels allows the sample recovery using commercially available kit and does not interfere with the extraction. Some recovery using this method with blue LED results in better quality compared to the SYBR Green I using transilluminator.