Team:Cambridge/Protocols/Buffers

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==Buffer Preparation==
==Buffer Preparation==
These buffers were used in various stages in purifying reflectin from inclusion bodies. The protocols have been adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here], and each makes a total of 100ml of buffer. All solutions should be stored at room temperature, but be aware that buffers containing urea will become increasingly basic over time.
These buffers were used in various stages in purifying reflectin from inclusion bodies. The protocols have been adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here], and each makes a total of 100ml of buffer. All solutions should be stored at room temperature, but be aware that buffers containing urea will become increasingly basic over time.
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:# Stir the solution until the powder is completely dissolved. Adjust the pH to 7.8 using 1M NaOH or 1M HCl.   
:# Stir the solution until the powder is completely dissolved. Adjust the pH to 7.8 using 1M NaOH or 1M HCl.   
:# Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.
:# Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.
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:# Store buffer at room temperature.  
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===Denaturing Binding Buffer (DBB)===
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:# To a 250ml beaker, add 0.58ml Stock Solution A, 9.42ml Stock Solution B, and 48.1g powdered urea.
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:# Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved.  Adjust the pH to 7.8 using 1M NaOH or 1M HCl. 
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:# Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.
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===Denaturing Wash Buffer (DWB)===
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:# To a 250ml beaker, add 7.38ml Stock Solution A, 2.62ml Stock Solution B, and 48.1g powdered urea.
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:# Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved. Adjust the pH to 6.0 using 1M NaOH or 1M HCl.
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:# Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.
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===Denaturing Elution Buffer (DEB)===
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:# To a 250ml beaker, add 10ml Stock Solution A and 48.1g powdered urea.
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:# Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved. Adjust the pH to 4.0 using 1M NaOH or 1M HCl. 
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:# Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.
===Safety===
===Safety===
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All safety measures relating to guanidine hydrochloride in solution from the [https://2011.igem.org/Team:Cambridge/Protocols/Buffers buffer protocol] apply here when dealing with GLB. In addition, all consumables and liquid waste that may have come into contact with the bacteria must be autoclaved before disposal.  
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Urea and guanidine hydrochloride are both skin and eye irritants. Lab coats, gloves and safety glasses should be worn at all times, and the chemicals should only be handled under a fume hood when in their powdered form. In case of contact with eyes or skin, flush immediately with running water - see the MSDS for each chemical for further details.
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Latest revision as of 20:31, 21 September 2011

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Contents

Buffer Preparation

These buffers were used in various stages in purifying reflectin from inclusion bodies. The protocols have been adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here], and each makes a total of 100ml of buffer. All solutions should be stored at room temperature, but be aware that buffers containing urea will become increasingly basic over time.

Stock Solutions

These were prepared for use in making the buffers that follow.

Stock Solution A

  1. Dissolve 2.76g of monobasic sodium phosphate and 29.29g of NaCl in 80 ml deionized water.
  2. Mix well and adjust the volume to 100ml with deionized water.

Stock Solution B

  1. Dissolve 2.84g dibasic sodium phosphate and 29.29g of NaCl in 80 ml of deionized water.
  2. Mix well and adjust the volume to 100ml with deionized water.

Guanidinium Lysis Buffer (GLB)

  1. To a 250ml beaker, add 0.58ml Stock Solution A, 9.42ml Stock Solution B, and 57.3g powdered Guanidine Hydrochloride.
  2. Stir the solution until the powder is completely dissolved. Adjust the pH to 7.8 using 1M NaOH or 1M HCl.
  3. Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.


Denaturing Binding Buffer (DBB)

  1. To a 250ml beaker, add 0.58ml Stock Solution A, 9.42ml Stock Solution B, and 48.1g powdered urea.
  2. Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved. Adjust the pH to 7.8 using 1M NaOH or 1M HCl.
  3. Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.

Denaturing Wash Buffer (DWB)

  1. To a 250ml beaker, add 7.38ml Stock Solution A, 2.62ml Stock Solution B, and 48.1g powdered urea.
  2. Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved. Adjust the pH to 6.0 using 1M NaOH or 1M HCl.
  3. Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.


Denaturing Elution Buffer (DEB)

  1. To a 250ml beaker, add 10ml Stock Solution A and 48.1g powdered urea.
  2. Stir the solution with gentle heating (50-60°C, do not overheat), gradually adding minimal amounts of de-ionised water until the powder is completely dissolved. Adjust the pH to 4.0 using 1M NaOH or 1M HCl.
  3. Bring the volume to 100 ml using de-ionised water and filter sterilize the buffer using a 0.45 µm filter.

Safety

Urea and guanidine hydrochloride are both skin and eye irritants. Lab coats, gloves and safety glasses should be worn at all times, and the chemicals should only be handled under a fume hood when in their powdered form. In case of contact with eyes or skin, flush immediately with running water - see the MSDS for each chemical for further details.

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