Question

Mary performs a PCR reaction and runs it on an agarose gel, using 100-bp ladder as her molecular weight standard. She images the gel using UV light and sees NO bands (no band in her PCR reaction and no bands for her ladder). Which of the following could explain these results? Explain each answer.

1. Mary forgot to add Gel Red when pouring the gel.

2. Mary's PCR product had an unusually high % of G and C bases.

3. Mary connected the electrodes to her gel incorrectly, swapping the positive and negative poles.

Answer 1

1.GEL RED is an intercalating nucleic acid stain used in molecular biology for agarose gel electrophoresis. Gel Red is structurally closely related to ethidium bromide and consist 2 ethidium subunits that are bridge by a linear spacer. when exposed to UV light , it will floresce with an orange clour that strongly intensifies after binding to DNA . if while pereforming PCR the staining agent is forgeten means , though the bands are produced it will not visbile .

2. In generally the PCR primer should have optimal G-C content should be 40-60%. G-C bonds contribute more stability ( increases the melting temperature) of primer or template binding than do A-T bonds. it has been reported that GC - rich region of the targeted DNA are difficult to amplify , so these region are generraly avoided when choosing a targeted DNA sequence.

3. if DNA is negitively charged , always move from a negitive feild to positive feild . the electrodes are desigined so that the negative is applied at the top of the gell where as the DNA is placed into the well . this causes the DNA to migreate down the lenth of the gel towards the +ve end . if you reveresed the electrodes, the electric feild will be reviresed . this would causes the DNA running in opposite direction . instead of migerating down the gel and seperating based on size the sample would migreate up and out the top of the gel right into buffer solution. because of this the band may not be appeared.