Team/CINVESTAV-IPN-UNAM MX/Systems.htm

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   <p><strong>27 July</strong></p>
   <p><strong>27 July</strong></p>
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  <p><strong>2 August</strong></p>
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     <li><strong>Party! all the systems are ready&hellip;</strong></li>
     <li><strong>Party! all the systems are ready&hellip;</strong></li>
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Latest revision as of 06:13, 26 October 2012

Rho

Systems assembly!

12 July

  • ZR Miniprep Kit from all  the genes
    • AppA
    • PpsR
    • PrrA
    • PrrB
    • PrrA Promoter
    • PpsR Promoter
  • Transformation of E. coli TOP10

13 July

  • Colony PCR
    • Taq Buffer                        5μL
    • MgCl2                             5μL
    • dnTP’s                             1μL
    • Forward                           1μL
    • Reverse                            1μL
    • Taq polymerase                0.5μL
    • H2O                                 36.5μL
      • TOTAL                 50μL

NEGATIVE

14 July

  • Culture from 4 colonies per gene. 6hrs
  • ZR Miniprep Kit
  • Agarose gel electrophoresis 1%
  • PCR from plasmid
    • Taq Buffer                       2μL
    • MgCl2                             2μL
    • dnTP’s                             0.5μL
    • Forward                          1μL
    • Reverse                            1μL
    • Taq polymerase                1μL
    • H2O                                 11.5μL
      • TOTAL                  20μL

POSITIVE

  • Band purification
  • Plasmid digestion  only  for genes (1.5h)
    • Xba1 and Pst1
  • Plasmid digestion  only  for promoters (1.5h)
    • EcoRI and SpeI
      • Agarose gel electrophoresis 1%
      • Genes ( +)
      • Promoters   (-)
  • Ligations
    • RBS+GENES
      • 20μL Reaction (see protocols)
  •  AGAIN!!!!  Plasmid digestion, only  for promoters (1.5h)
    • EcoRI and SpeI
      • Promoters   (-)
    • Agarose gel electrophoresis 4%
    • This was 4 more times!!!!!!!!!!!!!

15 July

  • Transformation by heat-shock

16 July

  • Ligation of promoters in psB1A3
  • Cultures of RBS+GENES (6hr)
    • ZR Miniprep kit

17 July

  • Digestion of RBS+GENS (1.5h, see protocols)
    • RBS+PrrA     S+P
    • RBS+PrrB     X+P
    • RBS+PrrC     E+S
    • DT                  E+X
    • RBS+AppA   X+P
    • Prom J23104         S+P
    • RBS+PpsR    E+S
  • Agarose gel electrophoresis 1%
    • POSITIVE (+)
  • Band Purification
  • Ligations A
    • 1A      RBS+PrrA +RBS+PrrB     
    • 2A      RBS+PrrC+DT       
    • 3A      Prom J23104+RBS+AppA
    • 5A      RBS+PpsR+ DT
  • Overnight cultures  of promoters+psB1A3

18 July

  • Transformation of ligations A
  • ZR Miniprep Kit
  • Digestiion
    • POSITIVE
  • Band purification
  • Ligation D
    • PrrA Promoter+GFP+DT
    • PpsR Promoter+GFP+DT

19 July

  • Transformation of ligations D
  • Cultures of ligations A (6h)
    • ZR Miniprep Kit
    • Digestion  (1.5h)
      • 1A      S+P    
      • 2A      X+P   
      • 3A      S+P
      • 5A      X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • Band purification
  • Ligations C
    • 1C      RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • 2C      Prom J23104+RBS+AppA+RBS+PpsR+ DT

20 July

  • Overnight cultures of ligations D (6h) (small colonies)
  • Transformation of ligations C

21 July

  • Cultures of Ligation C
    • Miniprep Kit
    • Digestion E and E+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
  • ZR Miniprep Kit of ligations D
    • Digestion E and X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • Band purification!
      • NEGATIVE
    • Overnight culture

22 July

  • ZR Miniprep Kit ligations D
    • Digestion E and E+P
      • Ppsr promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
      • POSITIVE +
  • Miniprep Kit ligation C
    • Digestion
      • PromJ23104                                                      S+P
      • RBS+PrrA +RBS+PrrB +RBS+PrrC+DT         X+P
      • Prom J23104+RBS+AppA+RBS+PpsR+ DT   S+P
    • Agarose gel electrophoresis 1%
    • High concentration! J
    • Band purification
      • POSITIVE +
    • Ligations E
      • 1E      PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
      • 2E      Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT

23 July

  • Transformation of ligations E
  • WE are also worked in the assembly of the butanol pathway + regulatory systems

24 July

  • 1E NOTHING!!!!!!!!!!
    • Again ligation!!
    • Miniprep Kit ligation C
    • Digestion
      • PromJ23104                                                         S+P
      • RBS+PrrA +RBS+PrrB +RBS+PrrC+DT            X+P
    • Agarose gel electrophoresis 1%
    • High concentration! J
    • Band purification
      • POSITIVE +
  • 2E; 3 COLONIES!!... ;       Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT
    • 6h culture!
    • ZR Miniprep Kit
    • Digestion (1.5h)    S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
    • Band purification
      • FIRST SYSTEM READY

25 July

  • Transformation of ligation 1E

26 July

  • 1E;  2 COLONIES!!! ; PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • 6h culture!
    • ZR Miniprep Kit
    • Digestion (1.5h)
    • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • JUST ONE MORE LIGATION
      • Overnight digestion
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27 July

  • ZR Miniprep Kit ligations 1D
    • Digestion X+P
      • PrrA promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
    • POSITIVE
  • 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
  • Ligation 2F:
    • PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT+ PrrA promoter+GFP+DT    

28 July

  • Transformation ligation 2F

29 July

  • NOTHING, AGAIN!           
  • ZR Miniprep Kit ligations 1D
    • Digestion X+P
      • PrrA promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
    • POSITIVE
  • 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
  • Ligation 2F

30 July

  • Transformation

1 August

  • 2F, 4 COLONIES    
    • 6h cultures
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • E and E+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
      • SECOND SYSTEM READY
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  • Party! all the systems are ready…

Biobricks Final assembly to psB1C3

    • From 3 of August to 15 of August, we did the following things.
      • Overnight and 6h cultures
      • ZR Miniprep Kit
      • Digestion (1.5h)
        • For ligate
        • For confirmation
      • Agarose gel electrophoresis 1%
      • Band purification
      • Ligation
      • Transformation; and again!
      • TOTAL                                  11 parts in psB1C3
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Rhodofactory 2012

icytdf
osli
bio
fermentAS
cinestav
genscript
unam
gto
quimica
valaner
ipn