IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/15

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Current revision (10:46, 16 July 2014) (view source)
(TesA/RBS Ligation Attempt 2)
 
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* Checked DNA concentrations
* Checked DNA concentrations
:TesA= 11.99 ng/μL ; RBS=32.99 ng/μL
:TesA= 11.99 ng/μL ; RBS=32.99 ng/μL
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*When divided by the number of base pairs: [TesA]=.018 strands/μL and [RBS]=.016 strands/μL
+
*When divided by the number of base pairs: [TesA]=.018 and [RBS]=.016
* Ligation
* Ligation
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{| {{table}} border="1" align=center <!-- Digest check rxn. table -->
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{| {{table}} border="1" align=center <!-- Ligation table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent (μL)
| bgcolor=#cfcfcf | Reagent (μL)
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|| &nbsp; || 10.0 μL --> 25°C/ ~1 Hr.
|| &nbsp; || 10.0 μL --> 25°C/ ~1 Hr.
|}
|}
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+
*After ligation, the mixture was heated to 65°C for 10 min. to denature the ligaze.
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+
*Transformation was then performed.
 +
:Three plates were grown overnight: RBS backbone (vector), TesA+RBS, and mCherryboth as a positive control and to replenish our stocks.
* Transformation
* Transformation
 +
:Unfortunately, no growth was seen on any of the plates, including the positive control of mCherry.
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TesA/RBS Ligation Attempt 2

  • Checked DNA concentrations
TesA= 11.99 ng/μL ; RBS=32.99 ng/μL
  • When divided by the number of base pairs: [TesA]=.018 and [RBS]=.016
  • Ligation
Reagent (μL) Volume (μL)
DNA(plasmid) 1.0 μL RBS, 2.0 μL TesA
10X buffer 1.0
dH2O 5.0
Ligaze 1.0
  10.0 μL --> 25°C/ ~1 Hr.
  • After ligation, the mixture was heated to 65°C for 10 min. to denature the ligaze.
  • Transformation was then performed.
Three plates were grown overnight: RBS backbone (vector), TesA+RBS, and mCherryboth as a positive control and to replenish our stocks.
  • Transformation
Unfortunately, no growth was seen on any of the plates, including the positive control of mCherry.
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