A cytobrush is a small brush that is traditionally used for performing pap tests. These are sterile and convenient for single usage inside the cheek of the mouth. Brushes are swirled in a solution of Chelex beads so that the chelating agents in the solution neutralize the action of divalent cations to protect the DNA. Placing these tubes of cells and Chelex onto a boiling heat block is designed to sterilize the solution for safe working conditions. An alternative to this use of Chelex is to displace the cells into a lysis buffer that will burst the cells and protect the DNA.
- Use sterile cytobrush and insert into mouth
- brush cytobrush on inside of cheek 25 times
- Swirl cytobrush in 100 μl of Chelex suspension (10% w/v) or lysis buffer containing EDTA
- Place centrifuge tube with Chelex and cell suspension on 100 °C heat block for 10 minutes
- Centrifuge tubes at maximum speed for 5 minutes
- DNA is in the supernatant. (avoid beads at bottom)
- Store DNA in -20 °C
PCR with PCR Beads
PCR Beads are prepared mixes of a thermostable polymerase with the appropriate salts (ions), buffering compounds and dNTPs that have been dehydrated into beads in PCR tubes. These beads require hydration with a primer solution and template DNA. Primer solution is usually prepared with sucrose or other dense compound with a dye molecule so that each PCR tube can be directly loaded onto an agarose gel.
- Add 22 μl of primer mix (forward and reverse) to beads
- Ensure that the the bead is dissolved
- Add 3 μl of DNA (if no amplification, try diluting the DNA sample at least 10x)