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

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(08/29/13)
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'''Golden gate'''
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'''Golden Gate'''
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* Repeat procedure from ###
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* Try to increase robustness by using more T4 ligase
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# hPCD + BL01 + pSB1A3
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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# pSB1A3 alone (neg ctrl)
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
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| rowspan="7" | [[Image:KAH011013_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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|-
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| DNA(plasmid) || 0.2 μL
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|-
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| primer 1 || 1.0
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|-
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| primer 2 || 1.0
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|-
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| 2x GoTaq || 25.0
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|-
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| dH<sub>2</sub>O || 22.8
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| &nbsp; || 50.0 μL
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|}
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PCR
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* 95°C, 3 min.
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
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* 72°C, 3 min.
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* 4°C, ∞
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Purification
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* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
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* Measure [DNA]
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Sample
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| bgcolor=#cfcfcf | OD 260
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| bgcolor=#cfcfcf | 260/280
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| bgcolor=#cfcfcf | ng/μL
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|-
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| 1. pSB1A3 || 0.126 || 1.888 || 77.357
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| 2. hPCD || 0.069 || 2.192 || 17.025
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| 3. BL01 || 0.121 || 1.898 || 66.878
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|}
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Dilutions: to final concentration of 20 fmol/μL
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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 || 6.7 || 13.3
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|-
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| hPCD || 186 || 17.0 || 2.8 || 17.2
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|-
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| BL01 || 2520 || 66.9 || 9.8 || 10.2
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* GG-PCR fragments generated on 1/10/13
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** Diluted to 20 fmol/μL
Golden Gate Reactions
Golden Gate Reactions
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Thermal cycling
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GG Thermal cycling
* [45°C, 2 min.; 16°C, 5 min.] x25
* [45°C, 2 min.; 16°C, 5 min.] x25
* 60°C, 20 min.
* 60°C, 20 min.

Revision as of 15:27, 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 generated on 1/10/13
    • Diluted to 20 fmol/μL

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

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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