Genotyping

Overview: During the first period of this lab students isolate genomic DNA from their cheek cells. During the second lab period, students finish cleaning up their DNA sample and amplify specific segments of their DNA using the polymerase chain reaction (PCR). During the third lab period, students electrophoretically analyze the results of the amplification on agarose gels.

Week Two

9:00 - Wash your hands. This lab requires no cross-contamination of DNA between students. Go get a bucket of ice for your table. Get your DNA prep from the freezer. Bring the sample to room temperature by warming it with your hands.

9:10 - Continue the DNA isolation by adding 200 uL of Protein Precipation Solution. Vortex at speed 6 for 20 seconds and then chill on ice for at least 5 minutes.

9:20 - Centrifuge your sample for 5 minutes at 16,000 x g. Remember to orient your tube so that you know where the pellet should be.

9:30 - While your sample is in the centrifuge, take a new 1.5 mL microcentrifuge tube, label it with your name or symbol, and put 600 uL of room temperature isopropanol into it.

9:40 - From the tube that was in the centrifuge, carefully add the DNA-containing supernatant to the 600 uL isopropanol in the new tube. Don't touch the pellet.

9:45 - Gently mix by inverting the tube about 30 times or until white threadlike strands of DNA form a visible mass. Often there are no strands, just tiny "floaties".

9:50 - Centrifuge your sample for 5 minutes at 16,000 x g. Remember to orient your tube so that you know where the pellet should be.

10:00 - Pipet out as much supernatant as you can without disturbing the pellet and discard the supernatant, perhaps into your old tube containing the protein. The tiny pellet is hopefully much more DNA than you will need. DON'T TOUCH THE PELLET!

10:05 - This time add 600 uL 70% ethanol and invert gently 30 times to wash the DNA pellet.

10:10 - Centrifuge your sample for 3 minutes at 16,000 x g. Remember to orient your tube so that you know where the pellet should be.

10:15 - Slowly and gently invert the tube onto a kimwipe backed by a paper towel to wick out the EtOH. Air-dry the pellet for at least 15 minutes. The pellet is very loose at this point and you must be carefull or you will ruin everything :-)    Remember it is just ethanol and not toxic. To dispose of the ethanol wet the paper with some water and throw the soaked paper in the trash.

10:40 - Add 100 uL of DNA Rehydration Solution and rehydrate the DNA by incubating at 65° C for 30 minutes.

10:40 - 11:10 Mix your warm wet DNA by gently tapping the tube every 10 minutes to help with the rehydration.

10:45 - Listen to Kibak for 3 x 8 minutes.

11:15 - Transfer 20 uL of your rehydrated DNA into a PCR tube.

11:16 - Mix in 20 uL of Master Mix by pipeting the solution up and down a couple of times.

11:20 - Load your tube into the PCR machine (Thermal Cycler) and let it cycle for about 3.5 hours.

We will freeze your PCR reaction for use next week. Save your remaining DNA in the freezer box.

Kibak or Beech will freeze PCR product when done... about 2:50 pm

Individual Researcher Materials

• Cheek cell DNA isolated during previous lab
• p1000 autopipet with tips
• p200 autopipet with tips
• p20 autopipet with tips
• PCR tube(s)
• capless holder tube(s)
• tube rack

• Master Mix (Keep on ice after adding primers and use within 30 minutes)
  • deoxynucleotides (dATP, dTTP, dCTP, and dGTP)
  • Taq DNA polymerase
  • buffer (50 mM KCl,10 mM Tris-HCl(pH 8.3), 1.5 mM MgCl2
  • Left Primer 5'-AACTGGGAAAATTTGAAGAGAAAGT-3'
  • Right Primer 5'-ATGGATGTAGTTGGTGTCATGGTCA-3
• microfuge
• thermal cycler