Team:Paris/August 26
From 2008.igem.org
(→Digestion of the PCR products) |
(→Ligation) |
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*transformation of TOP10 competent cells with 5 µL of ligation product | *transformation of TOP10 competent cells with 5 µL of ligation product | ||
*spreading on LB plates + ampicilline and incubation overnight at 37°C | *spreading on LB plates + ampicilline and incubation overnight at 37°C | ||
+ | |||
+ | |||
+ | ='''Construction of pFlhB - mRFP Tripart (LVA+)'''= | ||
+ | |||
+ | Aim : Construction of ''' "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
+ | We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth. | ||
+ | |||
+ | ==Digestion== | ||
+ | ===Digestion=== | ||
+ | [[Team:Paris/Notebook/Protocols#Digestion|Protocol Digestion]] | ||
+ | |||
+ | {| border="1" style="text-align: center" | ||
+ | |'''Name''' | ||
+ | |'''Template DNA''' | ||
+ | |'''Description''' | ||
+ | |'''Vol MP (µl)''' | ||
+ | |'''Vol H2O (µl)''' | ||
+ | |'''Enzymes''' | ||
+ | |- | ||
+ | |D187 | ||
+ | |MP168.1 | ||
+ | | RBS - mRFP - term (FV) | ||
+ | |9.00 | ||
+ | |15.7 | ||
+ | |EcoRI and XbaI | ||
+ | |} | ||
+ | |||
+ | ==='''Gel Verification'''=== | ||
+ | [[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]] | ||
+ | [[Image:KR000.JPG |thumb |Gel Verification of D187 digestion]] | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Well''' | ||
+ | |1 | ||
+ | |2 | ||
+ | |3 | ||
+ | |4 | ||
+ | |5 | ||
+ | |6 | ||
+ | |- | ||
+ | |'''Sample''' | ||
+ | |100 pb ladder | ||
+ | |MP168.1 | ||
+ | |no sample | ||
+ | |D187 | ||
+ | |colspan="2"|no sample | ||
+ | |- | ||
+ | |'''Expected size (pb)''' | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | colspan="2"| | ||
+ | |- | ||
+ | |'''Measured size (pb)''' | ||
+ | | | ||
+ | |style="background: #cbff7B"| | ||
+ | | | ||
+ | |style="background: #cbff7B"| | ||
+ | |colspan="2"| | ||
+ | |} |
Revision as of 17:47, 26 August 2008
Extraction of EnvZ* and OmpR* from E. coli genomeName of the PCR
ProtocolProtocol
PCR Programme
ResutsElectrophoresis settings
Results of the electrophoresis
Conclusion : All the PCR worked perfectly well ! Cleaning of the PCR productsThe cleaned PCR products are stored in the freezer overnight.
PCRPCR1
Program: Gradient Vf=20µL PCR2
Program: promoter Vf=50µL PCR3
Program: PCRFliA1 Program: PCRFliA2 Program: PCRFliA3
Cloning of EnvZ*Concentration measurement by Biophotometer
Ligation
Construction of pFlhB - mRFP Tripart (LVA+)Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth. DigestionDigestion
Gel Verification
|