BPS3151 Study Guide - Quiz Guide: Agarose Gel Electrophoresis, Amplicon, Plasmid

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ceriseshark335

30 Dec 2015

School

University of Ottawa

Department

Biopharmaceutical sciences

Course

BPS3151

Professor

Luc Poitras

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Laboratory class 2 assignment: only team #1 was able to digest the ptac-mat-tag-1 since its bands on the agarose gel picture match that of the digested ptac-mat-tag-1 (positive control done by the technicians). It is known that the supercoiled form of dna travels that farthest, the linear form of dna travels the second farthest, and the open circle form of dna travels the least far. The linear form of the plasmid corresponds to a double strand break and the circular form of the plasmid corresponds to a cleavage in one of the strands, resulting in a wider and relatively slower molecule in agarose gel. The band corresponding to team #3 somewhat resembles that of the undigested ptac-mat- Tag-1 (negative control done by the technicians) which remained supercoiled. This does not include the 6 base pairs of the recognition site on one primer nor the 6 additional base pairs in front of the recognition site on the same primer.

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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)

Evaluation of PCR product electrophoresed on a 0.8% agarose gel shows non-specific bands. The appropriate modification for the next PCR reaction is toa )increase the concentration of Taq polymerase.b) increase the number of PCR cycles.c) decrease the template denaturation temperature.d) reduce the concentration of primers.In the Sanger sequencing method, chain termination is the result ofa)misincorporation of dUTP instead of dTTP.b)incorporation of ddGTP.c)secondary structures in the template strand.d)sequencing through repeats.The purpose of EDTA in a DNA storage buffer is toa)maintain single strands.b)bind magnesiumc)activate DNases.d)dissolve DNA into solution.The thermal cycler ramp refers to thea)degree of incline of the machine on the laboratory bench.b)gradient between denaturation and annealing temperature.c)programming key to set number of PCR cycles.d)amount of time required to reach the desired temperature.Evaluation of a blood sample indicates the presence of a clot. Corrective action to salvage this specimen for analysis would most likely requirea)digestion with proteinase K.b)homogenizing the sample under UV irradiation.c)removal of the clot with urea.d)requesting a new sample immediately.Incorporation of biotin into template DNA by nick-translation requiresa)DNase.b)ligase.c)random hexanucleotides.d)3 temperatures.An isolated DNA sample is viscous, difficult to pipet, and will not resuspend in TE buffer after rehydration at 37C for 24 hours. The most appropriate step is toa)digest the DNA with a frequent cutting restriction enzyme.b)re-extract the original sample with higher volumes of reagents.c)increase the rehydration temperature to 95C.d)add more TE buffer.Excess glycerol in a restriction digestion reaction results ina)enzyme degradation.b)diminished enzyme activity.c)increased temperature required for digestion.d)decreased enzyme buffer pH.Primer dimers will most likely be a result of PCR whena)3' and 5' primers are designed as 50% GC.b)DNA template concentration is very low.c)enhancing agents are added to the master mix.d)template DNA is initially denatured completely.Correct operation of a pH meter requiresa)calculation of the electrode millivolt equivalents.b)standardization of the meter with a range of buffers.c)documentation of absorbance and standard deviation.d)verification of pH readings with calf thymus DNA.The uracil DNA glycosylase pre-PCR decontamination procedure failed. The most likely cause of the failure is thea)enzyme solution was heated prior to PCR.b)enzyme buffer solution was alkaline pH.c)N-glycosidic DNA bonds cleaved after enzyme treatment.d)PCR was performed with dTTP instead of dUTP.The real-time PCR analysis does not show the characteristic melting curve for the patient or control samples. The fluorimetry readings of the PCR products indicate the appropriate concentration of template was added to the real-time PCR reaction mix. Which of the following reagents was likely missing from the PCR master mix to account for this finding?a)SYBR Green Ib)template DNAc)DMSOd)5X Cresol RedWhich of the following conditions favor enhanced specificity of PCR?a)decrease in the concentration of dNTPsb)increase in the sodium chloride concentrationc)increase in the concentration of Taq polymerased)decrease in the ramp speed and temperatureWhich of the following compounds is an inhibitor of Taq polymerase?a)magnesiumb)hemoglobinc)DMSOd)betaineWhich of the following compositions of gels would allow for the efficient separation of linear DNA molecules in the range of 100 to 300 base pairs in length?a)0.3% agaroseb)1.0% agarosec)1.0% polyacrylamided)8.0% polyacrylamide4.92*10^4 cpm of a random primed 32P dCTP (3000 Ci/mmol) labeled oligonucleotide probe were precipitated from a 50 uL standard reaction by TCA and 5.28*10^4 cpm is the total cpm in the sample. What is the percent incorporation of radioactivity for this labeling reaction?a)1.86%b)46.50%c)93.00%d)98.40%The stock solution of HCl is 1M. How much stock solution is required to prepare 250 mL of 50 mM HCl?a)0.2 mLb)5 mLc)12.5 mLd)125 mLSpectroscopy measurement of a DNA sample isolated by salt extraction technique gave the following absorbance readings: A260 0.4032 A280 0.3765This sample is:a)acceptable for molecular testing.b)contaminated with salt.c)contaminated with protein.d)contaminated with high amounts of RNA.What is the dilution factor if 50 uL of blood is diluted with 950 uL of saline?a)19b)20c)40d)50Which of the following are used to prepare a 0.9% agarose gel?a)0.9 g agarose + 100 mL distilled waterb)0.9 g agarose + 100mL TAEc)9 g agarose + 100mL TBEd)90 g agarose + 100 mL bufferThe concentration of double-stranded DNA with an optical density reading of 1.0 isa)0.5 ug/ulb)1.0 ug/ulc)10 ng/uld)50 ng/ulHow much Tris base (MW 121.1) would be required to make 4 liters of Tris-EDTA buffer that is 15 mM Tris?a)0.7266 gramsb)0.1211 gramsc)1.8165 gramsd)7.266 grams

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BPS3151 Study Guide - Quiz Guide: Agarose Gel Electrophoresis, Amplicon, Plasmid

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