User:Matt Hartings/Notebook/Photosynthesis/2012/09/12

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(Objective)
Current revision (10:50, 12 September 2012) (view source)
 
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|style="background-color: #EEE"|[[Image:Hartings_AU_Photosynthesis_Lab_Header.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span>
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|style="background-color: #EEE"|[[Image:Hartings_AU_Photosynthesis_Lab_Header.png|128px]]<span style="font-size:22px;"> Protein Re-engineering</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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Objective

Run WT Hb expression with non iron protoporphyrin IX to see if expression proceeds as normal

Procedure

  1. Spin down starter cultures from the previous evening.
  2. Resuspend in 4mL fresh media
  3. Add 1mL of 100mg/ml ampicilin to each 2.8L fernbach flask
  4. Divide the 4mL resuspended cells among each expression flask
  5. Place flasks in shaker at 37C
  6. Grow to an OD of 0.6 at 600nm (Note: the cells grew to an OD of 0.9)
  7. Reduce temperature to 30C
  8. Add 1mL of 0.4M IPTG to each flask
  9. Add 1mL of metal-protoporphyrinIX to each flask
    1. 40mg of Mn-protoporphyrin IX in 1mL of DMSO
    2. 40mg of Ni-protoporphyrin IX in 1mL of DMSO (Note: I only had 16mg of this complex)
    3. 40mg of Co-protoporphyrin IX in 1mL of DMSO
    4. 40mg of Cu-protoporphyrin IX in 1mL of DMSO
  10. Shake for 4 hours after induction (I induced at 11:00am)


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