Apr 14, 2025
C. neoformans HMW DNA Extraction
This protocol is a draft, published without a DOI.
- Thomas Davies1
- 1University of Edinburgh
Protocol Citation: Thomas Davies 2025. C. neoformans HMW DNA Extraction. protocols.io https://protocols.io/view/c-neoformans-hmw-dna-extraction-d7w69phe
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: In development
We are still developing and optimizing this protocol
Created: April 14, 2025
Last Modified: April 14, 2025
Protocol Integer ID: 126654
Abstract
High molecular weight gDNA extraction protocol for C. neoformans cycling cultures. Suitable for downstream sequencing and PCR applications. Should yield 10-40µg gDNA per 50mL culture.
Materials
- Wash Buffer: 500mM NaCl, 50mM EDTA
- Spheroplasting Buffer: 1M sorbitol, 100mM Sodium Citrate (pH5.8), 10mM EDTA, 20mg/mL Sigma Lysing Enzyme (L1412, to be added immediately before use).
- Lysis Buffer: 100mM Tris (pH 7.5), 500mM NaCl, 100mM EDTA, 40µg/mL RNase A (to be added immediately before use).
- 2.5M KOAc in TE Buffer.
- 100% EtOH (Ice cold in freezer).
- 70% EtOH.
- Tris pH 8.
Pre-Culture Preparation
Pre-Culture Preparation
Grow approx 50mL C. neoformans culture to OD 1.5-1.7.
Centrifuge2000 x g, 4°C, 00:05:00 , remove supernatant.
Wash three times with 3mL wash buffer (0.5M NaCl, 50mM EDTA).
Centrifuge as above, remove supernatent and flash freeze in liquid nitrogen or dry-ice.
Spheroplasting
Spheroplasting
Thaw pellet at 37 °C , wash once in dH2O and centrifuge 2000 x g, 4°C, 00:05:00 .
Resuspend in 3mL sheroplasting buffer (1M sorbitol, 100mM Sodium Citrate (pH5.8), 10mM EDTA, 20mg/mL Sigma Lysing Enzyme (L1412)). Incubate in shaker 200 rpm, 37°C, 03:00:00
Centrifuge as above, remove supernatent and flash freeze in liquid nitrogen or dry-ice.
Lysis & DNA Extraction
Lysis & DNA Extraction
Thaw pellet at 37 °C , resuspend in 1.35mL lysis buffer (100mM Tris (pH 7.5), 500mM NaCl, 100mM EDTA, 40µg/mL RNase A), add 150µL 10% SDS. Incubate in shaker 200 rpm, 37°C, 01:00:00
Add 2mL 2.5M KOAc in TE, invert to mix, place in 15mL falcon tube and incubate On ice 00:05:00
Centrifuge2000 x g, 4°C, 00:05:00 , and transfer supernatant to new 15mL falcon. Repeat twice.
Transfer to 50mL falcon tube (approx 2.5mL per sample), add 6.5mL ice-cold 100% EtOH. Invert gently to mix and incubate On ice 01:00:00 at least.
Spit sample into 6x 1.5mL eppendorf tubes (should just fit), spin 14000 rpm, 4°C, 00:30:00 to pellet DNA.
Carefully remove supernatant and add 500µL 70% EtOH to remove salts from DNA, do not disturb pellet. Spin 14000 rpm, 4°C, 00:10:00 .
Carefully remove supernatant (from sides of tube too, two rounds of aspiration can help with this) and allow pellet to dry 00:05:00 at RT, longer times may result in dry pellet that is difficult to resuspent.
Add 20µL Tris pH 8 to each tube and incubate at least 00:30:00 to allow pellet to dissolve (can also pipette up and down gently, but I allow this time to let the pellet dissolve slowly). Gently pipette up and down to finish dissolving, and aliquot six samples back to 1 tube (120µL total).
Spin 14000 rpm, Room temperature, 00:30:00 to pellet any residual debris, and move supernatant to fresh eppendorf (low DNA binding tube ideally).
Can repeat once to increase purity.
Quantification of Yield
Quantification of Yield
Quantify DNA content via nanodrop and/or Qbit.
Nanodrop 260/280 ratio of 2.0 and 260/230 of 2.2 expected for high quality gDNA. Concentration quatification may be high compared to Qbit, which will provide the more accurate figure.
Acknowledgements
Adapted from Allshire group protocol, 2024.