Lab Notes-3/11/2010

Lab #3

Overview -
Looking for presence of 2 specific genes in Staphylococcus aureus (bacteria):
Pvl (Panton-Valentine Leukocidin) (Cid = to kill) (in= protein) (leuko = white blood cells) (leukocidin = protein that kills white blood cells). Gene could make you very sick
mecA (methicillin-resistance cassette A) Gene that makes the bacteria resistant to antibiotics (methicillin is an antibiotic).
If you have both genes, not only are you sick, but many antibiotics are useless to treat you.
(from our noses!)

Day #1 -

  • Get DNA out of S. aureus bacterial samples
    • Negative control = S. epi (labeled in blue)
    • Positive control = USA300 (labeled in pink)
  • Complete steps 1-7 from protocol (from Blake Hanson, Abby Harper, and Tara Smith of the University of Iowa College of Medicine)

Get DNA out of S. aureus bacterial samples (Protocol: Blake Hanson, Abby Harper, Tara Smith, U. of Iowa College of Medicine)
Negative control: Tube 1: S. epi (blue) strain with neither gene in it
Positive control: Tube 2: USA300 (pink) strain of Staphylococcus has both genes in it
3 experimental tubes with code numbers on them (Tube 5: 14 R 2007, Tube 3: 3 L 2007, Tube 4: 6 R 2007)
Step 1: centrifuge samples in large test tubes to make bacteria go to bottom of test tubes (to form pellet). Before it’s centrifuged, bacteria are everywhere in sample.
Step 2: Pour out supernatant into flask. Can dump supernatant out without losing pellet. Try to make pellet as “dry” as possible.
Step 3: we have 1 molar EDTA, but we need 50 mM EDTA. Need .5 mL of EDTA per sample. Need total of 2.5 mL of EDTA. Needed 0.5 mL of 1 M EDTA. Use a new pipet tip with each sample.
Step 4: Add the three “ingredients.” Use a new pipet tip with each sample. Pipet same up and down until sample is cloudy and resuspended.
Step 5: After incubation, put the total contents of each large tube into a small tube using a 1000 microliter pipet. Then, centrifuge for 2 min. Remove the supernatant by slurping it out with a pipet. Pellets are on same side as hinge. Slurp from opposite side of pellet. Do NOT disturb the pellet. If you do, you will have to centrifuge again.
Step 6: Add 600 microliters of Nuclei Lysis Solution. Either resuspend solution using Vortex (vibrator) or pipet (slurp solution up and down into pipet tip). Try to break up pellet, but solution doesn’t have to be 100% cloudy.
Step 7: Place samples in 80 C degree water bath for 5 min.
Step 8: put them in the freezer for next lab.

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