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Electrophoresis Synthesis Lab Report

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Method for Electrophoresis:

• For separation of Nucleic A. fragments of specific sizes an agarose solution is prepared in an electrophoresis buffer at an appropriate concentration.

• The mixture is heated in boiling water bath or microwave till agarose dissolves. Bottle cap must be kept loose.

• Transfer flask to water bath at 55°C using insulating gloves. Once melted gel has cooled, add ethidium bromide (final conc. 0.5 μg/ml). Swirl and mix.

• Position and appropriate comb of appropriate size, 0.5-1.0 mm over the plate to form complete well defined wells.

• The pour the warm agarose solution on the mold.

• The gel will polymerize at room temperature in 20-40 mins. Pour electrophoresis buffer on the gel and remove comb. Pour …show more content…

• Load size standards into slots on either sides of the gel.

• Close the lid of the tank.

• The gel is then subjected to an electrical field at a voltage of 1-5 V/cm and DNA will migrate towards the anode (red lead). For correctly attached leads: bubbles are generated at electrodes, bromophenol blue migrates from tank to walls. The electric field must be applied till bromophenol blue and xylene cyanol FF have migrated to a third of the gel.

• Once DNA/dye samples have separated enough to be detected, the electric current is switched off and the leads are taken off the tank. Alternatively, the gel can be stained by soaking it in a buffered 1:10,000- fold dilution of SYBR Green stock solution.

• DNA in agarose gels or Nucleic acids are visualized under 300-nm UV light after appropriately staining using either ethidium bromide or SYBR Green. A commonly used method to visualize DNA in agarose a gel is ethidium …show more content…

Albeit, SYBR Green has been preferred for single stranded (SS) nucleic acids d/t better comparative fluorescent yield than the former.

• According to Sambrook et al, fluorescent yield of SS nucleic A depends of binding of the dye to short intra-strand duplexes.

• SYBM Green increases detection sensitivity of DNA by 10-20 fold. This process utilizes yellow or green gelatin or cellophane filter recorded on a camera at an illumination of 300-nm UV light.

Applications of Electrophoresis of Nucleic Acid:
• Agarose Gel Electrophoresis is employed in restriction mapping of cloned DNA i.e. to estimate the size of DNA fragments digested with restriction enzymes.

• It has application in molecular genetics & genetic fingerprinting of PCR products.

• It is used to separate genomic DNS (Southern Blot) and RNA (Northern Blot).

• It’s used to resolve circular DNA with different supercoiling topology and to resolve fragments that differ due to DNA synthesis. The is reduction of DNA damage due to cross-linking proportionally with electrophoretic DNA migration.

• Agarose gel aids purification of DNA fragments which is necessary for a number molecular techniques such as

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