Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Shake-flask fermentation and detection of the target product

LX Liping Xu
PL Pingping Liu
ZD Zhubo Dai
FF Feiyu Fan
XZ Xueli Zhang
request Request a Protocol
ask Ask a question
Favorite

Yeast cells grown overnight were used to inoculate a 100 mL flask containing 15 mL of YPD medium (2% w/v glucose) to an initial OD600 of 0.2 and cultivated at 30 °C and 250 rpm for 10 days. The cells were then collected by centrifugation (6000g, 5 min), washed twice with sterile water, resuspended in cell lysis buffer (25 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA), and finally broken with glass beads (0.5 mm) using a BeadBeater mill (BioSpec Products, Bartlesville, Oklahoma, USA). The cell lysates were extracted with acetone: methanol (1:1) for squalene extraction. The organic phase was used for compound detection after filtering through a 0.22 µm pore-size Nylon 66 membrane (Millipore Corporation, Billerica, MA, USA). Squalene was detected by gas chromatography (GC) with an inlet temperature of 300 ℃, a sample volume of 1 µL, no shunt, solvent delay 12 min; Chromatographic column: hp-5 ms (30 m × 0.25 mm × 0.5 m); Chromatographic conditions: 80 ℃, 1 min; 20 ℃ min−1 to 300 ℃ for 18 min.

Copyright and License information: The Author(s) ©2021
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A