Freeze & Thaw lysis with XNA
- spin down bacteria, take 5uL of pellet, add to 20uL of XNA Extraction Solution
- Freeze in -20 for 5 min
- Thaw for a minute, room temperature or in your fingers
- repeat freeze – thaw two more times
- add XNA Dilution solution
General bacterial cell lysis
- For bacterial samples take a toothpick and scrape the teeth, or swab the throat, ears or between the toes. Resuspend material in 500ul of water. Freeze and thaw sample three times with vigorous shaking or vortexing between repetitions to break the bacterial cell wall. Although not all DNA will be released from the cells, there will be a sufficient quantity for PCR.
- Place the sample in a 95oC heating block, or in boiling water, for 5 minutes. This step inactivates the DNase molecules that are found in the sample preparation. If left intact, DNase could clip the desired DNA template molecule into fragments which would be unsuitable for PCR. If there is very little DNA in the sample preparation, the DNA can be concentrated by ethanol precipitation. The sample is now ready for PCR
Actinomycete DNA Extractions
- Mycelium was collected from plates and suspended in 500 μL of cetyltrimethylammonium bromide buffer (Hillis et al. 1996) in microcentrifuge tubes.
- Cells were broken by cycles of freezing and thawing (they were dipped in liquid nitrogen and then placed in a heat block at 65 °C); this procedure was repeated at least 3 times.
- One volume of chloroform was added, tubes were vortexed briefly, and then centrifuged for 10 min at 10 000g.
- Precipitation with 1 volume of 100% isopropanol was carried out overnight at –20 °C.
- After centrifugation, the DNA pellet was washed twice with 70% ethanol and air dried. DNA was resuspended in 50 μL of buffer TE/10 (10 mmol Tris–HCl/L, 0.1 mmol EDTA/L; pH 8.1); aliquots were diluted (1:200) in ddH2O and used for PCR amplification (Cafaro & Currie, 2005).