Sample information

They were moving very slow/sluggish but they were surrounded by other insects along with their kind. They were found in the rotten log. For physical characteristics, they have a yellow- cream color, wingless, brown head/mouth, six legs.

Methods

PCR Procedure:

1. Remove all unnecessary items from your lab station
2.  Put on nitrile gloves and clean all surfaces by wiping down with 70% Ethanol.
3.  Collect a 1.5 mL microcentrifuge tube . Label the tube “A” (for Arthropod PCR Cocktail). Place the tube in a 1.5mL tube rack.
4. Collect six 0.2 mL PCR tubes . Number and label them with your initials. Place the tubes in a PCR tube rack and record putative identification of each arthropod in the table.
5.  Use a P-2 or P-20 pipette to add 2 μL of template DNA to each corresponding Arthropod PCR tube above. It is critical that you change tips between each tube.
6.  Set aside the PCR tubes for now. Place the template DNA tubes back into storage. You will need these again for the Wolbachia PCR
7. Use a P-200 pipette to add each of the following reagents (from the “Total for 7 reactions” column) to the 1.5 mL tube marked “A”. Change tips between each reagent, and check off each reagent after it is added.
8. Vortex for 5 sec
9. Place the A tube on one side of the mini-centrifuge and the B (balancer tube) on the opposite side. This is called “balancing” the rotor; if the tubes are not balanced, it will make a loud sound and could damage the microcentrifuge. Quickly spin down  (~ 3-5 seconds) the Arthropod PCR Cocktail to collect liquid at the bottom of the tube.
10. Use a P-200 pipette to add 23 µL of the Arthropod PCR Cocktail (A) to tubes 1-6  (which should already contain the template DNA). Change tips between each tube. Never place a used pipette tip into the PCR Cocktail because it will contaminate all downstream reactions.
11. Tightly secure the lids on each tube.
12. Change the rotor  on your mini-centrifuge to the PCR tube rotor. Center tubes 1-3 on one side and 4-6 on the other side to balance the rotor. Briefly spin  to collect all liquid at the bottom of the tubes.
13. Transfer the tubes to the thermal cycler. Once everyone has placed their samples in the thermocycler, the program can be started  with the Arthropod PCR protocol. If using MiniOne, you may observe the PCR program as it cycles.
14. Repeat all the steps for Wolbachia PCR
15. Clean up your lab station and wipe surfaces with ethanol
16. When the thermal cycler is done, store samples in the refrigerator (4°C).

Gel Electrophoresis Protocol:

1.  Prepare the Gel with the DNA stain
2. Once the gel is ready, place the combs into it and let it cool down
3. Once the gel is cooled down and the wells are properly made within the gel, begin pipetting the samples
4. Pipette 10uL of the DNA ladder in the first well. Hover your pipette above the well, and slowly empty your pipette
5. Continue in this manner, carefully pipetting 10uL of each sample/sample loading buffer mixture into separate wells in the gel. Change tips between each sample
6. Place the lid on the gel box, connecting the electrodes appropriately
7. Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on size of the electrophoresis chamber
8. Let the power run until the yellow (or bottom) band in the loading dye is ¾ down the gel. Then, turn off the power, disconnect the electrodes, and remove the lid and the gel using gloves.
9. Place the gel on the transilluminator and note the presence/absence of bands within each well.

Results

I feel not that confident on my results because based on the gel electrophoresis results, my samples did not appear to have arthropod DNA and Wolbachia DNA. Therefore, there may have been an experimental error when conducting the procedures, not making it confident that my arthropods don’t have Wolbachia. However, based on some of the classmates I asked who has same/similar arthropods as me, they all said they didn’t appear to have Wolbachia.