User:Matt Hartings/Notebook/Photosynthesis/2013/02/12

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(Hb extraction)
Current revision (15:07, 12 February 2013) (view source)
(Hb extraction)
 
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Nyousha is making buffers for FPLC: 1L of 10mM Tris 50mM NaCl pH 8.5, 1L of 10mM Tris 1M NaCl pH 8.5
Nyousha is making buffers for FPLC: 1L of 10mM Tris 50mM NaCl pH 8.5, 1L of 10mM Tris 1M NaCl pH 8.5
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The buffers were filtered after they were made.
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Current revision

Protein Re-engineering Main project page
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DNA Gel

Maddie ran several PCR runs last week to try to mutate K31C Asc Hb to K31C T109C Asc Hb.

I made an agarose gel (1% - 0.25g agarose in 25mL TAE Buffer)

The lanes in the gel are

  1. Ladder
  2. WT
  3. F1
  4. F2
  5. F3
  6. F4

(each lane consists of 5uL DNA and 1uL loading buffer).

Results

Lane 2 showed nothing. Lanes 3-6 were really streaky. Potentially many different products, or perhaps too concentrated. Should run again more dilute.

Hb extraction

Last week we expressed WT Asc Mn-Hb and Ni-Hb. I am going to extract the Mn-Hb today.

  1. Sonicate: 30 seconds on, 30 seconds off, 3x
  2. balance in oak ridge tubes to within 0.01g
  3. centrifuge for 2 hours, 4C, at 18000rpm

Nyousha is making buffers for FPLC: 1L of 10mM Tris 50mM NaCl pH 8.5, 1L of 10mM Tris 1M NaCl pH 8.5

The buffers were filtered after they were made.


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