User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10

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Dilutions
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{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
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|- valign="top"
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| bgcolor=#cfcfcf | DNA
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| bgcolor=#cfcfcf | length (bp)
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| bgcolor=#cfcfcf | ng/μL
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| bgcolor=#cfcfcf | Vol.
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| bgcolor=#cfcfcf | + dH<sub>2</sub>O
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|-
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| pSB1A3 || 2000 || 77.4 || 7.7 || 12.3
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|-
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| hPCD || 186 || 17.0 || 2.1 || 17.8
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|-
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| BL01 || 2520 || 66.9 || 11.7 || 8.3
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|}
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* Golden Gate Reactions
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# hPCD + BL01 + pSB1A3
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# pSB1A3
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
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|- valign="top"
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| Reagent || 1 || 2
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|-
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| gg2 pSB1A3 || 1.0 || 1.0
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|-
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| gg3 hPCD || 1.0 || ---
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|-
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| gg4 BL01 || 1.0 || ---
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|-
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| 10x ligase buffer || 1.0 || 1.0
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|-
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| NEB T4 lgase || 0.25 || 0.25
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|-
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| BsmBI || 0.5 || 0.5
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|-
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| dH<sub>2</sub>O || 5.25 || 7.25
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|-
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| &nbsp; || 10.0 || 10.0
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|}
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Thermal cycler
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* [45°C, 2 min.; 16°C, 5 min.] x25
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* 60°C, 20 min.
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* 80°C, 20 min.

Revision as of 01:01, 11 January 2013

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

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


Dilutions

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 7.7 12.3
hPCD 186 17.0 2.1 17.8
BL01 2520 66.9 11.7 8.3


  • Golden Gate Reactions
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.



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