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

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* 4°C, ∞
* 4°C, ∞


* Measure [DNA]
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Sample
| bgcolor=#cfcfcf | OD 260
| bgcolor=#cfcfcf | 260/280
| bgcolor=#cfcfcf | 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
|}




Revision as of 21:58, 10 January 2013

Karmella'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, ∞
  • 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



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