Oct 21, 2021
DMSO stock preparation
- 1IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: Akashdutta 2021. DMSO stock preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bzbup2nw
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: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54356
Abstract
Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4oC. Long-term storage of bacteria is best at temperatures around -80oC. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol or DMSO (anti-freezing agents) is added to the media to store samples at such temperatures.
From the streaked plate of the bacteria, pick one colony and inoculate into a 250 mL flask containing 100 mL culture.
Grow the culture till it reaches OD∼ 0.6-0.8
Take the 100 mL culture and transfer 40 mL each into two falcon tubes.
Leave the remaining 20 mL culture in the flask. To that, add 80 mL of micronutrient enriched BG-11 media.
Take the two falcon tubes and centrifuge them at 5000 rpm, 25°C, 00:07:00 .
7m
Discard the supernatants of the centrifuged pellets.
Add 2 mL of BG-11 into each of the emptied falcon tubes. Use a 1 mL micropipette to mix such that the pellet is resuspended into a concentrated culture.
Add 140 µL of 7% DMSO.
Take 4 cryovials. Add 1 mL of the liquid in each cryovial.
Flash-freeze the vials using liquid nitrogen.
Store the stocks at -80 °C