User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09

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<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Golden Gate assembly: hPCD + BL01 + pSB1A3
* Golden Gate assembly: hPCD + BL01 + pSB1A3
 +
* Violacein: plates from Tabor; incubate o/n
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* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
-
Dilutions
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* Dilutions
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | DNA
-
| bgcolor=#cfcfcf | length
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| bgcolor=#cfcfcf | length (bp)
| bgcolor=#cfcfcf | ng/μL
| bgcolor=#cfcfcf | ng/μL
| bgcolor=#cfcfcf | Vol.
| bgcolor=#cfcfcf | Vol.
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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.
 +
* 80°C, 20 min.

Current revision

Karmella's BioBrick Cloning Main project page
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01/09/13

  • Golden Gate assembly: hPCD + BL01 + pSB1A3
  • Violacein: plates from Tabor; incubate o/n


Golden Gate Assembly

  • Use purified, measured PCR products from 1/05/13
  • Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
  • Dilutions
DNA length (bp) ng/μL Vol. + dH2O
gg2 pSB1A3 2000 67.7 7.7 12.3
gg3 hPCD 186 22.5 2.1 17.8
gg4 BL01 2520 55.9 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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