Team:Cambridge/Protocols/Buffers

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=Buffer Preparation=
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__NOTOC__
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==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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===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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:# Equilibrate the GLB to 37°C.
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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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:# Resuspend the cell pellet in the GLB.
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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===
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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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Revision as of 18:02, 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 [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here], and each make a total of 100ml of buffer.

Guanidinium Lysis Buffer (GLB)

  1. Make 8ml Guanidinium Lysis Buffer (GLB) via the appropriate protocol.
  2. Equilibrate the GLB to 37°C.
  3. Harvest bacterial cells from a 50 ml culture by centrifugation at 3,000 x g for 5 minutes.
  4. Resuspend the cell pellet in the GLB.
  5. Slowly rock the cells for 5–10 minutes at room temperature to ensure thorough cell lysis.
  6. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.
  7. Centrifuge the lysate at 3,000 x g for 15 minutes to pellet the cellular debris.
  8. Transfer the supernatant lysate to a fresh tube. Store on ice or at -20°C.

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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