Haynes Lab:Notebook/Engineering PC-TFs/2013/02/02

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
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|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #800000" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Summary==
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==2/2/2013==
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*  
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* '''Perform BsmBI/ T4 ligase mediated assembly'''
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* BsmBI cuts the DNA fragments and creates complementary overhangs.
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* Complementary sticky ends anneal via base pairing.
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* T4 ligase seals gaps in the phosphodiester DNA backbone.
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{| {{table}}
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| bgcolor="grey" | Reagent
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| bgcolor="grey" | Vol.
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| rowspan=7 | '''Thermal cycling'''
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* [45°C, 2 min.; 16°C 5 min.] x25
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* 60°C, 10 min.
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* 80°C, 20 min.
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* 4°C, ∞
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|-
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| 20 fmol (1 μL) of each DNA part || up to 8.0
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|-
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| 10x T4 ligase buffer (Promega) || 1.0
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|-
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| T4 ligase (NEB) || 0.25
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|-
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| BsmBI || 0.5
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|-
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| dH<sub>2</sub>O || 0.25
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|-
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| &nbsp; || 10.0 μL
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|}
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
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|- valign="top"
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| bgcolor="grey" | Reagent
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| bgcolor="grey" | 1
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| bgcolor="grey" | 2
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| bgcolor="grey" | 3
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|-
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| gg2 pSB1A3 || 1.0 || 1.0 || 1.0
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|-
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| gg3 hPCD || 1.0 || 1.0 || 1.0
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|-
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| BL02 || 1.0 || --- || ---
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|-
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| BL03 || --- || 1.0 || ---
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|-
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| BL04 || --- || --- || 1.0
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|-
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| 10x ligase buffer || 1.0 || 1.0 || 1.0
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|-
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| NEB T4 lgase || 0.25 || 0.25 || 1.0
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|-
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| BsmBI || 0.5 || 0.5 || 0.5
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|-
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| dH<sub>2</sub>O || 5.25 || 5.25 || 5.25
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|-
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| Total|| 10.0 || 10.0
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|}
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2/2/2013

  • Perform BsmBI/ T4 ligase mediated assembly
  • BsmBI cuts the DNA fragments and creates complementary overhangs.
  • Complementary sticky ends anneal via base pairing.
  • T4 ligase seals gaps in the phosphodiester DNA backbone.
Reagent Vol. Thermal cycling
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
20 fmol (1 μL) of each DNA part up to 8.0
10x T4 ligase buffer (Promega) 1.0
T4 ligase (NEB) 0.25
BsmBI 0.5
dH2O 0.25
  10.0 μL
Reagent 1 2 3
gg2 pSB1A3 1.0 1.0 1.0
gg3 hPCD 1.0 1.0 1.0
BL02 1.0 --- ---
BL03 --- 1.0 ---
BL04 --- --- 1.0
10x ligase buffer 1.0 1.0 1.0
NEB T4 lgase 0.25 0.25 1.0
BsmBI 0.5 0.5 0.5
dH2O 5.25 5.25 5.25
Total 10.0 10.0



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