Lab Protocol for PTC Genotyping Lab

This protocol is a continuation of the DNA isolation and PCR lab.  We will start with the 200 µL PCR tubes containing our amplified DNA.

Digestion of PCR Products with Restriction Enzyme Hae III

• Use a P20 micropipette set at 1 µL (010 top to bottom) to measure Hae III enzyme.

• Lift PCR tube so you can watch the liquid exit the pipet tip into the pink liquid.

• Remove pipet tip and attach to P200 micropipette set at 20 µL.

• Slowly pipet the pink liquid up and down 3 times to mix (avoid bubbles).

• Place tubes into thermal cycler set at 37 degrees C for a 30-minute incubation (digestion of PCR product).

Prepare and Load Agarose Gel with Digested PCR Products

• During 30-minute incubation, prepare 100 mL of 1.5% agarose in TBE running buffer and pour warm liquid (not hot) into electrophoresis chamber. We will prepare one solution and use it to pour 3 gels.

• Weigh 1.5 g agarose and put into 250 mL Erlenmeyer flask.

• Measure 100 mL TBE running buffer and pour into flask.

• Use microwave to completely dissolve agarose being careful that it does not boil over.

• When liquid is cool enough to handle, instructor will measure 5 µL EtBr (a carcinogen) and mix with agarose solution and pour approximately 1/3 of mixture into each of three electrophoresis  trays with 2 combs and allow gel to set (approximately 15 minutes).

• Pour TBE running buffer into electrophoresis chamber such that it covers gel.

• Set P20 micropipette to 16 µL. Practice loading middle wells of gel with 16 µL loading buffer.  Everyone should load a well. Two tables will load samples into the same gel.
• Load agarose gel with PCR samples (Time sensitive). Once the first sample is loaded, all samples should be loaded as quickly as possible to reduce the amount of diffusion prior to turning on the electricity.
• With P20 micropipette set at 16 µL, measure Hae III cut PCR sample and carefully load into an unused well that is on the left side of the tray. We will need to keep track of whose sample is loaded in each well.
• Measure 10 µL of size ladder into central well of agarose gel.
• Assemble the cover of the electrophoresis chamber with electrodes and turn on power.
• Run agarose gel for approximately 30 minutes.

Determine Phenotype and Genotype of Each Individual

• Instructor will turn off electrophoresis, transfer gel to documentation system, and program system to visualize EtBr-stained nucleic acids.
• Students can use their phone to take photographs of the agarose gel results (genotype determination).
• Everyone will test their ability to taste the chemical PTC. Three phenotype categories should be recognized: strong taster, weak taster, non-taster (phenotype determination).
• Each group should prepare a figure on which the source of DNA for each lane of the gel is labeled. The sizes of all DNA fragments in the size ladder and experimental samples should also be labeled.
• Each group will construct a table on which the genotype of everyone in the group will be indicated next to a column that indicates their phenotype.

Determine Cellular Location of PTC Using the Web

• Go to the following web page: https://wlab.ethz.ch/protter/#
• In the “please enter a UniProt protein accession:” box, type Q86UK4_HUMAN. We are entering the amino acid sequence of the human PTC protein into the search.
• Then click the submit button to obtain a diagram depicting the location of the protein in the cell.