Team:HokkaidoU Japan/Notebook/September28
From 2010.igem.org
(Difference between revisions)
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*PCR of E.coli with T3SSsignal and of GFP-double terminator | *PCR of E.coli with T3SSsignal and of GFP-double terminator | ||
- | = | + | = Ligation of plasmid and GFP-double terminator & Transformation = |
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant | #added 2 uL TE into plasmid solvant and GFP-double terminator solvant | ||
#mixed the samples | #mixed the samples | ||
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#plated the sample on LBA medium | #plated the sample on LBA medium | ||
#incubated at 37C | #incubated at 37C | ||
+ | |||
+ | = PCR of Parts = | ||
+ | |||
+ | *We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs. |
Revision as of 13:24, 27 October 2010
- glycerol-stock of E.coli with salmonella's BAC library vector
- making competent cell of E.coli with SPI2
- plasmid & GFP-double terminator's Ligation & Transformation
- PCR of E.coli with T3SSsignal and of GFP-double terminator
Ligation of plasmid and GFP-double terminator & Transformation
- added 2 uL TE into plasmid solvant and GFP-double terminator solvant
- mixed the samples
- added 5 uL Mighty mix
- incubated at 16C for 30min
- added the sample to 100 uL competent cell
- incubated at 0C for 30min
- heatshocked at 42C for 60sec
- incubated at 0C for 5min
- added sample to 400 uL LB
- incubated at 37C for 2 hours
- plated the sample on LBA medium
- incubated at 37C
PCR of Parts
- We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.