User:Jose Fabricio Lopez/Notebook/Logbook/2011/08/19: Difference between revisions

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=Objectives=
=Objectives=
* See resctriction in gel to perform ligation.
* See resctriction in gel to perform ligation.
* First PCR with standard primers for Isothermal assembly.


==Gel==
==Gel==
Line 20: Line 21:
[[User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/08/19| Ligation]] performed by [[User:Eduardo Vladimir Munoz|Vladimir]]
[[User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/08/19| Ligation]] performed by [[User:Eduardo Vladimir Munoz|Vladimir]]


==PCR with Standard primers for isothermal assembly==
Using [http://2010.igem.org/Team:Cambridge/Gibson/Introduction Cambridge protocol] for primers and backbone assembly.
{| border=1
!Reagents
!Insert of P1A7
!Insert of P1A7
!Backbone pSB17 P1A7
!Backbone pSB17 P1A7
![[User:Jose Fabricio Lopez/Notebook/Logbook/2011/07/06| pUXDNA ]]Insert Control
![[User:Jose Fabricio Lopez/Notebook/Logbook/2011/07/06| pUXDNA ]]Backbone Control
|--
|Buffer Phusion HF 10x
|10μL
|10μL
|10μL
|10μL
|10μL
|10μL
|--
|8mM dNTPs
|5μL
|5μL
|5μL
|5μL
|5μL
|5μL
|--
|Primer Fwd
|5μL
|5μL
|5μL
|5μL
|5μL
|5μL
|--
|Primer Rev
|5μL
|5μL
|5μL
|5μL
|5μL
|5μL
|--
|DNA
|1μL
|1μL
|1μL
|1μL
|1μL
|1μL
|--
|Phusion
|.5μL
|.5μL
|.5μL
|.5μL
|.5μL
|.5μL
|--
|H<sub>2</sub>O
|23.5μL
|23.5μL
|23.5μL
|23.5μL
|23.5μL
|23.5μL
|--
|Total
|50μL
|50μL
|50μL
|50μL
|50μL
|50μL
|}




Revision as of 18:11, 22 August 2011

iGEM 2011 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Objectives

  • See resctriction in gel to perform ligation.
  • First PCR with standard primers for Isothermal assembly.

Gel

  • Lane 1 Ladder O'Gene Ruler mix
  • Lane 2 B0015 EcoR1/Pst1
  • Lane 3 pSB1T3

It is a little bit dirty but usefull for Ligation.

Ligation performed by Vladimir


PCR with Standard primers for isothermal assembly

Using Cambridge protocol for primers and backbone assembly.

Reagents Insert of P1A7 Insert of P1A7 Backbone pSB17 P1A7 Backbone pSB17 P1A7 pUXDNA Insert Control pUXDNA Backbone Control
Buffer Phusion HF 10x 10μL 10μL 10μL 10μL 10μL 10μL
8mM dNTPs 5μL 5μL 5μL 5μL 5μL 5μL
Primer Fwd 5μL 5μL 5μL 5μL 5μL 5μL
Primer Rev 5μL 5μL 5μL 5μL 5μL 5μL
DNA 1μL 1μL 1μL 1μL 1μL 1μL
Phusion .5μL .5μL .5μL .5μL .5μL .5μL
H2O 23.5μL 23.5μL 23.5μL 23.5μL 23.5μL 23.5μL
Total 50μL 50μL 50μL 50μL 50μL 50μL




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