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Team/CINVESTAV-IPN-UNAM MX/Systems.htm
From 2012.igem.org
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
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
2 August
- 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
Rhodofactory 2012
