User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29

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# hPCD + BL01 + pSB1A3
# hPCD + BL01 + pSB1A3
# pSB1A3 alone (neg ctrl)
# pSB1A3 alone (neg ctrl)
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* GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL
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* GG-PCR fragments generated on 1/10/13
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Golden Gate reactions for today
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** Diluted to 20 fmol/μL
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-
 
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Golden Gate Reactions
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# hPCD + BL01 + pSB1A3
# hPCD + BL01 + pSB1A3
# pSB1A3
# pSB1A3

Revision as of 16:29, 29 August 2013

Karmella's BioBrick Cloning Main project page
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08/29/13

  • Golden Gate Assembly optimization - increase ligase amount; redo hPCD assembly



Golden Gate

  • Try to increase robustness by using more T4 ligase
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3 alone (neg ctrl)
  • GG-PCR fragments were generated on 1/10/13 and diluted to 20 fmol/μL

Golden Gate reactions for today

  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

GG Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞




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