McClean: ConA

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(Materials)
(Procedure)
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==Procedure==
==Procedure==
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Mix 2.5mls of the CaCl2 and the MnCl2 solution in 45mls of water (50ml total volume).
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Mix 2.5mls of the CaCl<sub>2</sub> and the MnCl<sub>2</sub> solution in 45mls of water (50ml total volume).
Check the pH of this solution.  It should be between 6-7.  Do not pH this solution with NaOH as the manganese will precipitate out.
Check the pH of this solution.  It should be between 6-7.  Do not pH this solution with NaOH as the manganese will precipitate out.
In this mixture, dissolve a 20mg/ml solution of conA. Dilute from this mixture down to 2mg/ml in miliQ water.  Store the 2mg/ml aliquots at -20°C, thaw just before microscopy.  Use the 2mg/ml solution to prime flow cells.
In this mixture, dissolve a 20mg/ml solution of conA. Dilute from this mixture down to 2mg/ml in miliQ water.  Store the 2mg/ml aliquots at -20°C, thaw just before microscopy.  Use the 2mg/ml solution to prime flow cells.

Revision as of 18:02, 2 November 2011

Contents

Overview

This is the concanavalin A solution we use for "sticking" yeast cells to the coverslip during microfluidics experiments. Generally, we make a large stock and store it in the -20°C freezer in ~ 500μL aliquots. These can be removed from the freezer and thawed at room temperature when needed. For most applications, the thawed stock can be left at room temperature for several days (or even freeze-thawed several times) before it's efficiency is reduced noticeably.

Materials

  • 1M CaCl2 solution in sterile H2O
  • 1M MnCl2 solution in sterile H2O
  • Concanavalin A (MP Biomedicals, #150710)

Procedure

Mix 2.5mls of the CaCl2 and the MnCl2 solution in 45mls of water (50ml total volume). Check the pH of this solution. It should be between 6-7. Do not pH this solution with NaOH as the manganese will precipitate out. In this mixture, dissolve a 20mg/ml solution of conA. Dilute from this mixture down to 2mg/ml in miliQ water. Store the 2mg/ml aliquots at -20°C, thaw just before microscopy. Use the 2mg/ml solution to prime flow cells.

Final concentrations: 5mM MnCl2 5mM CaCl2 2mg/ml concanavalin A

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Megan N McClean 17:59, 2 November 2011 (EDT) The solution can be stored at room temperature after the initial thaw for up to a week without noticeable loss of efficacy. I have also put a solution of conA through several -20°C freeze thaws without noticing a significant change in "sticking power".

Contact

or instead, discuss this protocol.


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