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1 Jun 2018

I recently performed a series of experiments using the enzyme acid phosphatase. To observe the inhibition of enzyme activity, disodium phosphate was added to an experimental solution, and the rate of activity was compared to the control, in which the enzyme performed under standard conditions (pH 5/20C). The solution with the addition of disodium phosphate performed poorly when compared to the control, as expected. My question is, what kind of inhibition is this? Competitive or noncompetitive? And why?

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2 Jun 2018

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Design an experimentin which you will test the effect of an acidic fluid on enzymaticactivity. (Recall: enzymes are proteins.) To complete this project,it may be useful for you to review the Scientific Method Tutorial (found the CourseContent section of the classroom under the Science Learning Centerlink) and the Scientific Method lab (Lab 1), so that you canbetter understand how to design an experiment. It may also behelpful for you to review Lab 4 (Enzymes). As you review Lab 4, youwill be reminded that there are several factors that impactenzymatic activity: pH, temperature, and amount of reagent. Feelfree to refer to observations and information from Lab 4 as youcomplete the questions required in the Final Applied Project (seethe questions below).

As you design yourexperiment for this project, please remember that you are trying toexamine how an acidic fluid will modify the outcome of an enzymaticreaction. To successfully complete this project, you will need toidentify the question(s) being asked in your experiment and thehypothesis that you are testing. In your experimental design, youmust clearly explain what you are doing. That means that you willneed to identify the enzyme and the acid, as well as explain yourexperimental protocol (this information will help you to answerquestion 2). You must alsothoroughly explain how the addition of the acidic fluid impactedthe overall reaction process (this information will help you toanswer question 4).

Hint: Keepin mind that the acid will change the environmental conditions ofthe experiment (for example, a low pH value could change the shapeof the active site on the enzyme protein), without directlyparticipating in the reaction.

please address the following:

1. What question are you asking?Clearly state your hypothesis.

2. Design an experiment. Provide adetailed account of the materials and methods used to conduct theexperiment. Also include the methods for data collection andanalysis.

3. Conduct the experiment and recordyour results below. What did you observe? Which samples showedbubbling?

4. Use your knowledge about factorsthat effect enzymatic activity to interpret your results. It may benecessary for you to refer to your lab manual and/or otherresources. What effect did the acid have on the enzyme?

5. Was your hypothesis supported? Whatis your conclusion? Explain your answer.

6. Cite all references used tocomplete your Final Applied Project.

scarletsardine134

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

crimsoncaribou758

Evaluation of PCR product electrophoresed on a 0.8% agarose gelshows non-specific bands. The appropriate modification for the nextPCR reaction is to

a )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 resultof

a)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 to

a)maintain single strands.

b)bind magnesium

c)activate DNases.

d)dissolve DNA into solution.

The thermal cycler ramp refers to the

a)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 mostlikely require

a)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-translationrequires

a)DNase.

b)ligase.

c)random hexanucleotides.

d)3 temperatures.

An isolated DNA sample is viscous, difficult to pipet, and willnot resuspend in TE buffer after rehydration at 37C for 24 hours.The most appropriate step is to

a)digest the DNA with a frequent cutting restriction enzyme.

b)re-extract the original sample with higher volumes ofreagents.

c)increase the rehydration temperature to 95C.

d)add more TE buffer.

Excess glycerol in a restriction digestion reaction resultsin

a)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 when

a)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 requires

a)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 procedurefailed. The most likely cause of the failure is the

a)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 characteristicmelting curve for the patient or control samples. The fluorimetryreadings of the PCR products indicate the appropriate concentrationof template was added to the real-time PCR reaction mix. Which ofthe following reagents was likely missing from the PCR master mixto account for this finding?

a)SYBR Green I

b)template DNA

c)DMSO

d)5X Cresol Red

Which of the following conditions favor enhanced specificity ofPCR?

a)decrease in the concentration of dNTPs

b)increase in the sodium chloride concentration

c)increase in the concentration of Taq polymerase

d)decrease in the ramp speed and temperature

Which of the following compounds is an inhibitor of Taqpolymerase?

a)magnesium

b)hemoglobin

c)DMSO

d)betaine

Which of the following compositions of gels would allow for theefficient separation of linear DNA molecules in the range of 100 to300 base pairs in length?

a)0.3% agarose

b)1.0% agarose

c)1.0% polyacrylamide

d)8.0% polyacrylamide

4.92*10^4 cpm of a random primed 32P dCTP (3000 Ci/mmol) labeledoligonucleotide probe were precipitated from a 50 uL standardreaction by TCA and 5.28*10^4 cpm is the total cpm in the sample.What is the percent incorporation of radioactivity for thislabeling reaction?

a)1.86%

b)46.50%

c)93.00%

d)98.40%

The stock solution of HCl is 1M. How much stock solution isrequired to prepare 250 mL of 50 mM HCl?

a)0.2 mL

b)5 mL

c)12.5 mL

d)125 mL

Spectroscopy measurement of a DNA sample isolated by saltextraction technique gave the following absorbance readings: A2600.4032 A280 0.3765

This 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 with950 uL of saline?

a)19

b)20

c)40

d)50

Which of the following are used to prepare a 0.9% agarosegel?

a)0.9 g agarose + 100 mL distilled water

b)0.9 g agarose + 100mL TAE

c)9 g agarose + 100mL TBE

d)90 g agarose + 100 mL buffer

The concentration of double-stranded DNA with an optical densityreading of 1.0 is

a)0.5 ug/ul

b)1.0 ug/ul

c)10 ng/ul

d)50 ng/ul

How much Tris base (MW 121.1) would be required to make 4 litersof Tris-EDTA buffer that is 15 mM Tris?

a)0.7266 grams

b)0.1211 grams

c)1.8165 grams

d)7.266 grams

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