Jun 01, 2023
Cryptococcus neoformans DNA Extraction Method
- 1Duke University
Protocol Citation: Amy Gladfelter 2023. Cryptococcus neoformans DNA Extraction Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2522jl1y/v1
Manuscript citation:
Based on Pitkin et al. (1996) Microbiology 142: 1557-1565. Results in ~0.5-2 mg DNA.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2018
Last Modified: June 01, 2023
Protocol Integer ID: 17443
Abstract
Extraction method to obtain genomic DNA from Cryptococcus neoformans.
Attachments
Guidelines
Extraction buffer (100 ml)
| Stock Solution | Add | Final Concentration | |
| 1 M Tris-HCl, pH 7.5 | 10 ml | 100 mM | |
| 5 M NaCl | 14 ml | 0.7 M | |
| 0.5 M EDTA | 2ml | 10 mM | |
| CTAB powder | 1g | 1% | |
| B-mercaptoethanol (14 M) | 1ml | 1% | |
| Water | 73 ml |
CTAB is mixed alkyltrimethyl ammonium bromide, Sigma cat.# M7635. This takes time to go into solution. You can also use solid NaCl rather than a 5 M solution if that is easier. The buffer lasts four- six months at room temperature.The buffer seems to work better for long strands and spooling if β-mercaptoethanol is added just prior to use.
Safety warnings
See SDS (Safety Data Sheet) for hazards and safety guidelines.
Grow a 50 mL YPD culture overnight (16:00:00 ), shaking at 30 °C .
Pellet cells in tabletop centrifuge in a 50 ml disposable tube.
Note
Optional: wash pellet with water and repeat spin.
Freeze cells at -20 °C to -80 °C for <30 min, then dry in a freeze drying machine.
Add the equivalent of 3 mL to 5 mL of 2 mm glass beads and vortex/shake until the cell pellet is broken and a fine powder is created.
In fume hood, add 10 mL CTAB extraction buffer (see Guidelines) and mix.
Incubate at65 °C for 00:30:00 .
In fume hood, add 10 mL chloroform and gently mix for approximately 00:01:00 .
Spin in a table top centrifuge for 00:10:00 (2,500 – 3,000 rpm).
Remove supernatant (c. 7 ml) and add to an equal volume of isopropanol in a 15 ml disposable tube.
Gently rock back and forward to mix.
If the DNA precipitates in strands and clumps, spool out with a glass pipette and transfer to eppendorf containing 1 mL 70% ethanol.
Otherwise, spin in a table top centrifuge for 00:10:00 , pour off supernatant and use 1 mL 70% ethanol to wash DNA pellet and transfer it to an eppendorf tube.
Spin sample in microcentrifuge for 5-10 minutes. Remove ethanol and allow to air dry.
Resuspend DNA in either water or TE buffer (c. 500 μl).
RNase can be added to final concentration of 20 μg/ml if needed.
Run 1 µL on an agarose gel to check concentration and quality.