2) When plasmid DNA is isolated and run on an agarose gel, why do you observe 2, 3 or even 4 bands? Please discuss.
3) You used a commercial plasmid mini-prep kit to extract plasmid DNA. Please discuss the principle behind the DNA extraction.
4) In one of the DNA extraction labs, you used a nanodrop spectrophotometer. a) What is a nanodrop spectrophotometer? b) What does the A260/A280 ratio mean? c) What is a good A260/A280 ratio for DNA? d) If your DNA has a A260/A280 ratio value of 1.3, what does it mean? e) If your DNA has a A260/A280 ratio value of 1.9, what does it mean?
5) You used PCR to amplify a region from the Amelogenin gene. Please discuss the principle of PCR. List some applications of PCR.
6) You also used gel electrophoresis for analyzing the PCR products and the plasmid DNA. Please discuss the principle of gel electrophoresis with a figure.
2) plasmid tends to remain in multiple forms unless it is digested or enzymatically treated. it acquires superhelical form, linear form, or relaxed circular form. these three forms have different properties of mobility. the superhelical form moves faster in a gel, while circular form moves slower. if the plasmid has a nick in one its strands, it gives an additional band.
3) plasmid mini-prep kits are based on alkaline lysis principle.
the bacterial pellet is treated EDTA, which disrupts the cell wall of the bacteria and destabilizes the phosphate backbone of DNA.
alkaline solution (NaOH) consisting of detergent (SDS) is added. the detergent denatures cell wall, and the alkali denatures both chromosomal DNA and plasmid DNA.
potassium acetate is added to acidify the solution and renature the plasmid DNA but not chromosomal DNA since it got precipitated out of the solution.
4. a) nanodrop spectrophotometers can measure sample volumes as small as 0.5 uL.
b) A260/280 ratio is used to check the purity of the isolated DNA.
c) for DNA, A260/280 value of 1.8 or higher indicates pure DNA (without protein contamination)
d) if the DNA has an A260/A280 value of 1.3, it indicates protein contamination.
e) A260/A280 value of 1.9 indicates pure DNA.
2) When plasmid DNA is isolated and run on an agarose gel, why do you observe...