Sep 02, 2020

Public workspaceTAP media preparation V.2

This protocol is a draft, published without a DOI.
  • 1Ronin Institute
Joao Vitor Molino
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External link: https://www.chlamycollection.org/methods/media-recipes/hutners-trace-elements/
Protocol Citation: Joao Vitor Molino 2020. TAP media preparation. protocols.io https://protocols.io/view/tap-media-preparation-bjmhkk36Version created by Joao Vitor Molino
Manuscript citation:
Molino JVD, Carvalho JCMd, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. PLoS ONE 13(2): e0192433. doi: 10.1371/journal.pone.0192433
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: August 12, 2020
Last Modified: September 02, 2020
Protocol Integer ID: 40329
Keywords: Microalgae, Recombinant, electroporation, plasmid, Media, Algae, Chlamydomonas reinhardtii
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Abstract
This protocol describes the preparation of TAP media. Usually used for growing algae cells, as Chlamydomonas reinhardtii. The protocol is derived from the protocol descrived at https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/.

[Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669]
Guidelines
All steps described in this protocol are intended to be conducted in a research laboratory.
Safety warnings
Use EPIs at all times.
Concentrated acetic acid solution used during preparation.
Before start
  • Prepare stock solutions
  • Separate flasks to distribute the prepared media for autoclavation
  • Separate magnetic stirrer for mixing
  • Large enough becker for media preparation
  • Pippetes/volume measuring apparatus for components

Components mixing
Components mixing
  1. Add approximatelly Concentration90 % volume of ddH20 to a large enough vessel.
  2. Add a magnetic bar and start and keep mixing with a magnetic stirrer during the preparation.
  3. Place pH probe electrode in the solution for pH monitoring


For Amount1 L final media volume

  1. Add Amount10 mL 2M Tris base (e.g. Trizma)
  2. Add Amount10 mL Solution A
  3. Add Amount1 mL Phosphate solution
  4. Add Amount1 mL Hutner`s trace solution
  5. Add Amount1 mL Glacial acetic acid , then add drops until Ph7 is reached
  6. Stop mixing, and add dd H20 until Amount1 L final volume is reached
  7. Start mixing again until complete mixing is achieved. (5 minutes should suffice).

Componets concentration and informations.
Stock solutionComponentAmount for Stock (g) *Check final volume for each in column AMolecular weight (g/mol)Final media concentration (mM)
Solution A (1000ml)NH4Cl4053.497.48
MgSO4 . 7H2010246.470.406
CaCl2 . 2H2O5147.010.34
Phosphate solutionK2HPO427174.20.620
(250mL)KH2PO414136.0860.41
Tris Solution (1000mL)Tris242.28121.1420
Acetic acidAcetic Acid Glacial60.05~17.5

Hutner`s trace composition and protocol for preparation can be found here.
Liquid transfer
Liquid transfer
  1. Transfer the newlly prepared media to flasks for autoclavation

Typically:

  • 1L flasks with blue caps
  • Erlenmeyers with a max volume capacity of 100 mL are filled with 50 mL, capped with alluminium foil (2 layers)
  • Erlenmeyers with a max volume capacity of 500 mL are filled with 250 mL, capped with alluminium foil (2 layers)



Autoclavation
Autoclavation
  1. Place flasks inside the autoclave (For flasks with lid, make sure it is loosen enough to allow vapor passage)
  2. Set autoclavation to Temperature121 °C for at least Duration00:15:00 , 15 psi.
  3. After autoclavation, wait media to cool down and it is ready for use.