Haynes Lab:Notebook/Engineering PC-TFs/2013/02/02: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
|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>}}
|style="background-color: #800000" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==Summary==
==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.
{| {{table}}
| bgcolor="grey" | Reagent
| bgcolor="grey" | Vol.
| rowspan=7 | '''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
|-
| dH<sub>2</sub>O || 0.25
|-
| &nbsp; || 10.0 μL
|}
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
| bgcolor="grey" | Reagent
| bgcolor="grey" | 1
| bgcolor="grey" | 2
| bgcolor="grey" | 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
|-
| dH<sub>2</sub>O || 5.25 || 5.25 || 5.25
|-
| Total|| 10.0 || 10.0
|}
 




Latest revision as of 22:24, 26 September 2017



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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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