Encyclopedia of Experiments: Cancer Research
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Transcript
- Environmental enrichment, or EE, refers to the modifications in the housing parameters to obtain beneficial effects on various diseases such as cancer. In a tumor bearing mouse, EE activates the immune system and reduces inflammation, which ultimately improves the gut microbiota and improves survival rate. To study the changes in gut microbiota, add the desired amount of stool lysis buffer in a tube containing the mouse stool.
Centrifuge the sample and transfer the supernatant to a fresh microcentrifuge tube. Add guanidinium chloride lysis buffer that denatures DNase and RNase. Next, add ethanol and transfer the sample to a silica spin column.
Ethanol helps DNA bind to silica. Centrifuge the sample. Now, transfer the column to a fresh microcentrifuge tube and add an elution buffer to the column to elute DNA.
Centrifuge the sample and discard the column. Transfer the sample into a PCR tube and run PCR to amplify the DNA. Load the DNA on agarose gel and perform electrophoresis to quantify the size of microbial DNA. In the following protocol, we will isolate DNA from stools to analyze the EE effect on colon microbiota in the tumorigenic mouse.
- Utilize a commercial kit to isolate microbial DNA from the stool following a stool pathogen detection protocol. When doing so, transfer the samples from the minus 80 degrees Celsius freezer on dry ice and weigh them. Next, set up a PCR that amplifies the microbial DNA as described in the text protocol. Then, clean up the reaction products using magnetic beads.
For the cleanup, first, briefly centrifuge the amplicon PCR plate to pull the reaction products. Next, vortex the magnetic beads to evenly disperse them and transfer 20 microliters aliquots to each reaction well. Mix the beads with the solution thoroughly by pipetting the entire volume slowly 10 times. Then, let the beads settle for 5 minutes.
Next, place the PCR plate on a magnetic stand and wait 2 minutes for the supernatants to clear following magnetic collection of beads. Then, remove and discard the supernatants. Next, with the plate still on the magnet, fill each well of beads with 200 microliters of fresh 80 percent ethanol and wait 30 seconds. Then, carefully remove the supernatant.
Repeat this wash step once and let the beads air dry for 10 minutes. Now, elute the reaction products from the beads by removing the plate from the magnetic stand and adding 52.5 microliters of Tris to each well. Completely mix the beads into solution by pipetting the entire volume slowly 10 times. Then, collect the beads on the magnetic stand as before and transfer 50 microliters of the supernatants to a clean PCR plate. Cover the plate with a clear adhesive and store it at minus 20 degrees Celsius for up to one week.
