Lab 4: Biochemistry II

Question 1

learning objectives

Answer 1

• > Perform an experiment to test the rate of reaction of an enzyme
• > Use a standard curve to convert from absorbance to molarity
• > Draw a flowchart of the steps required to isolate genomic DNA from processed foods and note the purpose of each step.
• > Match the ingredients in PCR master mix to the function of each component.
• > Diagram the cycling steps of the thermal cycler and match to temperature, reagents, and action.

Question 2

phosphate buffer

Answer 2

helps maintain constant pH level in a particular environment

Question 3

what is acetylthiocholine

Answer 3

In this lab, we will use commercially produced acetylthiocholine (ATCh) as the substrate for acetylcholinesterase activity instead of the natural substrate, acetylcholine (ACh). AChE hydrolyzes ATCh into acetic acid and thiocholine (lab 3)

Question 4

polymerase chain reaction

Answer 4

PCR technology makes it possible to amplify a specific region of a DNA molecule, for instance, a specific region in an organism’s genome. Using PCR, a scientist can prepare a gene for cloning; detect gene mutations, such as those that lead to cancer; monitor cancer therapy; and quickly detect bacterial and viral infections. PCR makes it possible to detect the presence of bacteria or other species that cannot be cultured or identified.

• > this method requires an ezyme called DNA polymerase
• > DNA polymerase attaches to single DNA strands that become templates for adding successive nucleotides to the growing molecule.
• > One common heat-stable DNA polymerase is Taq polymerase from the bacterium Thermus aquaticus.

Question 5

cycling in PCR

Answer 5

Exponential increases in the number of copies of DNA made in PCR are achieved in three basic steps in a cycle—denaturation, annealing, and extension

Question 6

PCR I

Answer 6

isolate dna from food

amplify dna from food

Question 7

why these steps for PCR

Answer 7

➢Grinding/homogenization of food to release DNA
➢InstaGene chelates divalent ions (e.g. Mg2+)
necessary for DNA degrading enzymes (e.g. DNases)
➢Only 50 μl of food transferred otherwise InstaGene is overwhelmed
(~ 5 mg of original material)
➢Boiling releases DNA from food into the InstaGene solution
➢Pellet InstaGene and food debris because InstaGene inhibits PCR
reaction (Taq needs Mg++)

Question 8

contents of PCR mastermix

Answer 8

●Primers
○Plant PSII primers or
       *GMO primers
●dNTPs
●Taq polymerase
●Buffer
●Mg 2+

Question 9

amplification of template DNA learning objectives

Answer 9

• > Match the ingredients in PCR master mix to the function of each component.
• > Diagram the cycling steps of the thermal cycler and match to temperature, reagents, and action.
• > Determine the outcome of a GMO detection procedure by PCR amplification using plant-specific and GMO specific primers, interpret AGE results from such amplifications, and troubleshoot errors.