Haynes Lab:Notebook/Engineering PC-TFs/2013/02/08: Difference between revisions

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(Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
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==Summary==
==Summary==
*  
* * '''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
|}
 




Revision as of 13:11, 9 February 2013

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Summary

  • * 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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