Student Genotyping

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

Week One

2:00 - Preparing agarose gels for use in Week Three.

1. Three gels must be prepared for Thursday section, two for Friday. Four lanes are used for standards and controls. The rest are open, so class has to calculate which comb to use.
2. Listen to Kibak instructions on how to pour and store agarose gels.
   
3. The gels consist of 1.5% agarose, so we will add 0.6 g low melting temp agarose to 40 mL 1xTAE buffer.
4. Using a clean ehrlenmeyer flask stuffed with a kimwipe, microwave the solution 20 seconds, then swirl and microwave another 10 seconds, repeating until completely dissolved but not boiling over.
5. When the agarose solution has cooled enough to hold the bottle but still hot, and while wearing gloves, add 18 uL Ethidium Bromide to the liquid agarose solution and swirl to completely disperse.
6. Pour evenly into the formed gel box with comb without hesitating.
7. Let solidifiy 30-60 minutes without disturbing.
8. Seal the gel and gel form in a plastic bag and keep in refrigerator until use.

2:30 - Listen to your instructor.  We have plenty of time today, so no hurry or panic required. You may wish to get a drink of water and rinse you mouth gently before this lab, but do not brush your teeth or use mouthwash.

2:35 - Wash your hands. This lab requires no cross-contamination of DNA between students.  As with the protein lab, these methods are extremely sensitive and can detect unimaginably small quantities of the correct DNA.

2:40 - Read through the protocol, ask questions and set up your lab notebook. 

Cheek Cell DNA Isolation Protocol: Step two on, you are on your own today. Cross off the numbers as you complete each step.

1. Get one ice bucket for each table.
2. Obtain a tube of EDTA and one of Nuclei Lysis Solution. Add 120 uL of the 0.5 M EDTA to 500 uL Nuclei Lysis Solution. Mix well and keep chilled on ice (solution should turn cloudy) for use in step 10.
3. Shake out a clear sterile 1.5 mL microcentrifuge tube and label it with your initials (at least 3 initials if possible).
   Do not use tape and do a good job because the writing has to survive long immersion in a hot water bath.
   
4. At least one hour after brushing, and while wearing gloves, obtain a cup and pour in 5-10 mL of a 3% sugar solution.
   
5. Vigorously swish the 3% sucrose solution around in your mouth while gently biting cheeks and otherwise mechanically dislodging cheek cells... continue agitating the solution in your mouth for 20-30 seconds.
   
6. Collect the "rinsate" in the cup (i.e. spit the solution back into the cup).
   
7. Swirl the rinsate in the cup so that the sloughed-off cheek cells do not settle, and pipet 1 mL of cheek cell suspension into your labeled sterile microcentrifuge tube.
   
8. Centrifuge the tube(s) at 3000 rpm for 10 minutes (orient the tubes so that you know where the pellet should be).
   
9. Without disturbing the pellet, pipet out the supernatant and discard. Leaving a little bit of supernatant in the tube is preferable to accidentally pipetting out some of the cells.
   
10. Add 600 uL of the chilled EDTA/Nuclei Lysis Solution to the remaining pellet of cells.
11. Vortex until the pellet is completely resuspended. This may take a while.
12. Add 17.5 uL of Proteinase K.
13. Incubate at least 5 hours at 55°C.
    
14. Kibak or Beech will store your labeled tubes in the -20°C freezer until you will use them next week.