Team:Cambridge/Protocols/Buffers

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==Buffer Preparation==
==Buffer Preparation==
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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 make a total of 100ml of buffer.
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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 buffers should be stored at room temperature, but be aware that the buffers containing urea will become increasingly basic over time.
===Guanidinium Lysis Buffer (GLB)===
===Guanidinium Lysis Buffer (GLB)===
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:# Make 8ml Guanidinium Lysis Buffer (GLB) via the appropriate [[Team:Cambridge/Protocols/Buffers | protocol]].  
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:# To a 250ml beaker, add 0.58ml Stock Solution A, 9.42ml Stock Solution B, and 57.3g powdered Guanidine Hydrochloride.
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:# Equilibrate the GLB to 37°C.  
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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.
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:# Harvest bacterial cells from a 50 ml culture by centrifugation at 3,000 x g for 5 minutes.  
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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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:# Resuspend the cell pellet in the GLB.
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:# Store buffer at room temperature.  
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:# Slowly rock the cells for 5–10 minutes at room temperature to ensure thorough cell lysis.  
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:# Sonicate the cell lysate on ice with three 5-second pulses at high intensity.  
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:# Centrifuge the lysate at 3,000 x g for 15 minutes to pellet the cellular debris.  
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:# Transfer the supernatant lysate to a fresh tube. Store on ice or at -20°C.
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===Safety===
===Safety===

Revision as of 18:14, 20 August 2011

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Buffer Preparation

These buffers were used in various stages in purifying reflectin from inclusion bodies. The protocols have been adapted from here, and each makes a total of 100ml of buffer. All buffers should be stored at room temperature, but be aware that the buffers containing urea will become increasingly basic over time.

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.
  4. Store buffer at room temperature.


Safety

All safety measures relating to guanidine hydrochloride in solution from the 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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