1.) You measure the OD600 of your culture of chloramphenicol-treated XL1-Blue/pT

Question

1.) You measure the OD600 of your culture of chloramphenicol-treated XL1-Blue/pTrc99A cells as described in Step 1 of Experiment 3A and obtain a value of 0.265. Assuming a plasmid copy number of 570 plasmids per cell, and a 100% recovery of plasmid DNA from 3.00 mL of the cells, what is the theoretical yield of plasmid DNA in micrograms (ug)? 2 points (Note: the size of pTrc99A is 4176 base pairs and the average molecular weight of double-stranded DNA is 660 g/mole of base pairs; report your answer with three significant figures) Tries 0/3

2.) Would you expect your actual yield of DNA to be more or less than the theoretical yield? 1 point Tries 0/1

3.) Assuming 100% recovery of DNA from the phage DNA isolation (Experiment 3B) and considering that not all the phage in your stock are able to infect E. coli successfully, would you expect your actual yield of DNA to be more or less than the theoretical yield calculated from your titer of the phage stock? 1 point Tries 0/1

4. Choose (True or False) whether each of the following parameters effects the rate of migration of DNA through agarose gels. 2 points Length of time in electric field DNA shape (supercoiled vs. relaxed circular vs. linear)

Volume of sample loaded (assuming all of the sample is contained within the loading well and enters the gel)

Electric field strength (volts per centimeter)

DNA size (molecular weight)

Physical length of the agarose gel

Polarity of the electric field Porosity of gel (% (w/v) agarose) Tries 0/3

5.) Ethidium bromide stains DNA by intercalating between adjacent base pairs, resulting in an increase in fluorescence, and can be used to quantify the mass of DNA present in a band of DNA on an agarose gel. Will the staining intensity of 100 ng of supercoiled plasmid DNA be the same as 100 ng of the plasmid that has been linearized by a single-site digestion with a restriction endonuclease?1 point Tries 0/1

6.) Choose (True or False) whether each of the following parameters effects the distance of migration of DNA through agarose gels. 1 point Polarity of the electric field

DNA size (molecular weight)

Length of time in electric field

Electric field strength (volts per centimeter)

Volume of sample loaded (assuming all of the sample is contained within the loading well and enters the gel)

Physical length of the agarose gel

Porosity of gel (% (w/v) agarose)

DNA shape (supercoiled vs. relaxed circular vs. linear)

Explanation / Answer

2. Yield of DNA is calculated from different methods, actual yield of DNA is obtained from measuring the obsorbance by adjusting turbidity. Theoretical yield of DNA is obtained by getting the amount of DNA products at the end of the reaction whereas actual yield is the one obtained by the amount of DNA fragments obtained from complete reaction of limiting fragments in the reaction.

4. Rate of DNA migration in agarose gel was affected by

DNA shape and size, polarity of electric field, porosity of gel, electric field strength

5. Factors affects distance of DNA in agarose Gel includes

Porosity of gel, volume of sample loaded, DNA size, physical length of agarose gel, DNA shape.

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