Lab Notes for 2nd Lab Report

March 11

DNA extractions
Looking for presence of 2 specific genes in Staphylococcus aureus (bacteria):
Pvl (Panton-Valentine Leukocidin) From wikipedia: Is the cause of necrotic lesions involving the skin or mucosa. (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) From Wikipedia: antibiotic. Have gene, you’re resistant to many antibiotics.
If you have both genes, not only are you sick, but many antibiotics are useless to treat you.

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
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 pour 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 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.
End of Steps for March 11th. Place samples in freezer.
March 18th
Defrost samples by holding them in your hand. Then, Vortex samples.
Step 8: Add 2 microliters of RNAase Solution. Invert tubes to mix (shake them side by side and up and down).
Step 9: Incubate samples for 30 min.
Step 10: Add Protein Precipitate Solution. Vortex for 20 sec.
Step 11: Incubate the sample on ice for 5 min.
Step 12: Centrifuge
Step 13/14: Add 600 microliters of isopropanol to five new “mini” test tubes. Label test tubes 1-5. Put the supernatant in the test tubes with the isopropanol. (The supernatant contains the DNA. The isopropanol makes the DNA form a pellet.) Invert test tubes until string-like strands of DNA are visible. Throw away the pellet which is in the previous test tubes.
Step 15: Centrifuge
Step 16: Poor off the supernatant and drain the tube on absorbent paper. (Tap the test tube pretty hard on the paper towel. Don’t let even a drop of liquid remain). Let the pellet dry for 10-15 min. The pellet contains the DNA.
Step 17: Add 100 microliters of DNA Rehydration Solution to test tubes. Put samples in freezer.
(March 25)
Step 18: Make agarose gel – 1 g agarose and 100 mL buffer. Microwave; pour gel into boat; put boat in fridge to cool off
Step 19: Mix 10 mL of each DNA sample and 5 mL loading dye on a piece of parafilm
Put 5 dots of loading dye on parafilm – make sure there’s plenty of space between dots
Pipet 10 mL of each DNA sample onto parafilm – pipet each DNA sample on an edge of a dot and then pipet up and down to mix
Step 20: Get gels out of fridge. Set up “machine.” DNA moves towards negative (red) end of machine. So, place boat with wells near black (positive) end of machine. Pipet samples into wells. Start up the electricity.

Step 21: Get ready to perform PCR while “machine” is running – need ntps, primers, and DNA polyermase (put these on ice) – bacteria DNA serves as our template DNA.
Step 22 – label 5 tubes while machine is running with sample numbers and group name
Step 23: Add 45 microliters water + buffer mixture to each tube (tube marked W)
Step 24: Add 1 microliters NTP mix to each tube (N)
Step 25: Add 2 microliters of each primer (forward and reverse) to each tube (F= forward, R=reverse)
Step 26: Add 2 microliters sample DNA to each tube
Step 27: Robson added 0.5 microliter of taq polymerase to each tube

Step 28: Put samples in centrifuge to ensure chemicals mix
Step 29: Robson placed samples in PCR machine – machine heated and cooled samples during 50 cycles

April 8, 2010
Use samples placed in PCR machine, not our old, original samples
5 microliters loading dye plus 15 microliters sample (probably can’t fit more than 25 microliters total (dye and sample) /well)
Also run Lambda DNA marker (10 microliters of marker plus 5 microliters of loading dye)
Pippet 6 dots of 5 mL loading dye onto parafilm
5 dots of loading dye are mixed with each sample and 1 dot of loading dye is mixed with 10 microliters of Lambda DNA marker
Place samples in wells of agarose gel – electrocute them
We added ethium bromide directly to the buffer before the gel was electrocuted instead of soaking the gel in ethium bromide like usual

muchas gracias
de nada
Thank you!
Is anybody done yet?

Anybody willing to share some of their sources for the background? We're having trouble finding some

We found 15. Try the bacteria name with the positive control under google scholar.

thank you! and no we are not done yet lol

we are doneish

Does anyone know when the lab tutors are there?

JPs there on Mon from 8-10 pm? and Weds 8-10 pm?

Alicia is for sure there on Weds from 10-12 pm and 4-6 pm.

tried to use the tutor…wasn't very helpful to our group and the other group there

Set up a time with Robson by texting her.

we set up a time with Robson too and she didn't show up.

She's sometimes in the lab.

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License