Sample information

Methods

• First the vile holding the arthropod was removed from the freezer and the insect was carefully transfer using tweezers to a petri dish 
• Next 5 ml of DI water was used to rinse the arthropod. Once rinsed, the water was dumped into the sink and the insect was placed back onto the petri dish. 
• Since the insect was smaller than a grain of rice, it was placed in a 1.5 ml microcentrifuge tube  
• Next 180 ul of Buffer ATL was added to the tube 
• Using a sterile pestle the arthropod was grinded for 2.5 minutes while hearing a squeaking sound 
• After being crushed, 20 ul of Proteinase K was added. Then 200 ul Buffer AL was added and immediately mixed via a vortex for 20 seconds. 
• Next the insect was incubated for 20 minutes. After the incubation, the arthropod was put in the freezer for 2 days. 
• After the insect was removed from the freezer, using a pipette set at 1000 microliters, the liquid was taken and put into a spin column. 

DNA Purification

• Once the liquid was collected, it was then released into a DNeasy Mini column, but was careful putting in the column without touching the membrane. 
• Then the whole groups DNeasy columns were centrifuged for 1 minute at 6,000 x g (8,000rpm). Then the spin column was taking out and the liquid was poured into the waste beaker. Once the liquid was out, the spin column was put back into the centrifuge tube. 
• This step was repeated for the negative and positive collections. 
• Then, each group member had 500 ul of Buffer AW1 added to the spin column. The Buffer AW1 was also added to the negative and positive columns. 
• Again, this was centrifuged for 1 minute at 6,000 x g (8,000rpm) 
• Once again, the spin column was taken out of the centrifuge tube and the liquid was discarded into the waste beaker.  
• Then 500 ul Buffer AW2 was added into the tubes, but instead centrifuged for 3 minutes at 17,000 x g (14,000rpm) 

DNA Elution

• The spin column was then put into a1.5 ml centrifuge tube. 
• 100 ul of Buffer Ae was then pipetted in directly into the spin column membrane. 
• Then the column was then left with the lid closed for 1 minute 
• After the room temperature incubation, the tube was centrifuged for a minute at 6,000 g  
• Once this was done, the spin column was discarded and the 1.5 ml tube was kept. At this point the DNA is in the 1.5 ml tube. 
• After the DNA was put into the freezer. 

Results

There is definitely a line on my specimen that represents that it is an arthropod. Therefore, I have high confidence that the insect is negative in Wolbachia bacteria.