IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/16

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(Autocreate 2014/07/16 Entry for IGEM:Arizona_State/2014/Notebook/asu_igem_2014)
Current revision (13:33, 16 July 2014) (view source)
(Ligation attempt # 3)
 
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==Entry title==
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==Ligation attempt # 3==
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* Insert content here...
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* Results from ligation #2 were inconclusive, as no growth was seen on the plates after transformation. Possible cause is that the competent cells were compromised somehow through the steps of the transformation and either died, or did not pick up DNA with chlor. resistance.
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*Today's work was to test the viability of yesterdays competent cells and re-run the ligation to see if we can get it to work.
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#Competent cells:
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#*Mixed 50 μL of competent cells, treated with the mCherry plasmid, with 500 μL of SOC and .5 μL of chloramphenicol, and incubated @ 32°C
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::After about 2 hours, no growth was seen.
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#*Poured remaining mixture of mCherry competent cells onto a new chlor. plate and incubated @ 32°C
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#Ligation
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#*Started a new ligation using the exact same measurements as yesterday (7/15) with the same positive control (mCherry) and negative control (RBS backbone).
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#*Changes: ligaze was not denatured in PCR
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#*Theoretically, today's competent cells are not compromised in any way and will grow properly

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Ligation attempt # 3

  • Results from ligation #2 were inconclusive, as no growth was seen on the plates after transformation. Possible cause is that the competent cells were compromised somehow through the steps of the transformation and either died, or did not pick up DNA with chlor. resistance.
  • Today's work was to test the viability of yesterdays competent cells and re-run the ligation to see if we can get it to work.
  1. Competent cells:
    • Mixed 50 μL of competent cells, treated with the mCherry plasmid, with 500 μL of SOC and .5 μL of chloramphenicol, and incubated @ 32°C
After about 2 hours, no growth was seen.
    • Poured remaining mixture of mCherry competent cells onto a new chlor. plate and incubated @ 32°C
  1. Ligation
    • Started a new ligation using the exact same measurements as yesterday (7/15) with the same positive control (mCherry) and negative control (RBS backbone).
    • Changes: ligaze was not denatured in PCR
    • Theoretically, today's competent cells are not compromised in any way and will grow properly


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