Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/18 May 2015"

(→‎Remaking of Starter Culture and Miniprep of Expression Vector)
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<a href="https://2015.igem.org/Team:UCLA"><li>HOME</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Team"><li>TEAM</li></a>
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<a href="#"><li>PROJECTS
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            <ul>
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<!-- <a href="https://2015.igem.org/Team:UCLA/Description"><li>Description</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Projects/Silks"><li>Silk Genetics</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Projects/Functionalization"><li>Functionalization</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Projects/Protein_Cages"><li>Protein Cages</li></a>
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<a href="#"><li>PARTS
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<a href="https://2015.igem.org/Team:UCLA/Parts"><li>Team Parts</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Basic_Part"><li>Basic Parts</li></a> 
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<a href="https://2015.igem.org/Team:UCLA/Composite_Part"><li>Composite Parts</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Part_Collection"><li>Part Collection</li></a> 
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</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Notebook"><li>NOTEBOOKS
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                                                <ul>
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics"><li>Spider Silk Genetics</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk"><li>Honeybee Silk</li></a> 
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Recombinant_Expression"><li>Recombinant Silk Functionalization</li></a> 
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Materials"><li>Materials  Processing</li></a> 
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Protein_Cages"><li>Protein Cages</li></a>
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                                                        <a href="https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study"><li>UCLA Measurement Interlab Study</li></a>
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</ul>
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                                        </li></a>
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<a href="#"><li>MEETING NOTES
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<a href="https://2015.igem.org/Team:UCLA/Notebook/General_Meetings"><li>General Meetings</li></a>
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                                                        <a href="https://2015.igem.org/Team:UCLA/Notebook/Coordinator_Meetings"><li>Coordinator Meetings</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Advisor_Meetings"><li>Advisor Meetings</li></a>   
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</ul>
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                                        </li></a>
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<a href="https://2015.igem.org/Team:UCLA/Attributions"><li>ATTRIBUTIONS</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Collaborations"><li>COLLABORATIONS</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Practices"><li>HUMAN PRACTICES</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Safety"><li>SAFETY</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Modeling"><li>MODELING</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Measurement"><li>MEASUREMENT STUDY</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Software"><li>SOFTWARE</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Entrepreneurship"><li>ENTREPRENEURSHIP</li></a>
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</ul>
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=5/18=
 
=5/18=

Revision as of 00:51, 28 May 2015


5/18

Remaking of Starter Culture and Miniprep of Expression Vector

  • Starter Culture from 5/17 had an OD 600 of 3.1 for tube #1 and 3.0 for tube #2
    • This is too high, as the desired range is around 0.5 to 1.5.
  • Remaking starter culture. Picked 3 colonies from plate #2 (P+S 2) and incubated in 3 tubes with 10 ml LB and 10 ul 1000X chloramphenicol
    • Incubated at 37C starting at noon. (200 rpm)
    • At 2 pm, the OD was still essentially 0.
    • So apparently, I was reading the spec incorrectly. I was using cuvettes, but I had forgotten to change the setting to "10 mm cuvettes"
    • At 7:30 pm, (7.5 hours of incubation), the OD600 was 1.21
      • This is a little too high, as 0.8 is about optimal, but I went with it anyways.
  • Added 5 ml of the starter culture to 245 ml Difco LB and 250 ul of 1000X chloromphenicol to a 1L flask with the little ridges in the bottom for better aeration (baffled flask)
  • Incubate at 37C 200 rpm starting at 8 pm
    • At 10:40pm OD600 = 0.336
    • At 11:05pm OD600 = 0.441
  • At this point I added 1 ml of 100 mM iptg to make 0.4 mM final IPTG concentration.
  • Placed back into the incubator starting at 11:10 PM

Redo of Pet24a Miniprep

  • When Megan did the miniprep from the pET 24a transformed bacteria, the yield was very low.
  • I re-picked 2 colonies from the pET 24a transformed plate I made on 5/10 and inoculated them on 5/17.
    • One tube didnt seem to have any growth and the other tube had very precipitated bacteria.
    • The yield of the miniprep from tube #2 was very low.

That plate from 5/10 seems funky, so Megan will re do the transformation, with less DNA and will make a 1:10 dilution plate as well