Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10"

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* 4°C, ∞
 
* 4°C, ∞
  
 +
 +
Purification
 +
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
 
* Measure [DNA]
 
* Measure [DNA]
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| 3. BL01 || 0.121 || 1.898 || 66.878
 
| 3. BL01 || 0.121 || 1.898 || 66.878
 
|}
 
|}
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Revision as of 22:00, 10 January 2013

Owwnotebook icon.pngKarmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers



Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)



Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar



Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878