Team:HokkaidoU Japan/Notebook/September28
From 2010.igem.org
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+ | *PCRed according to the table below.98C and 68C were 45 cycles. | ||
+ | {|style="text-align: center" class="protocol" | ||
+ | !temp | ||
+ | !time | ||
+ | |- | ||
+ | |94C | ||
+ | |2 min | ||
+ | |- | ||
+ | |98C | ||
+ | |10 sec | ||
+ | |- | ||
+ | |68C | ||
+ | |60 sec | ||
+ | |- | ||
+ | |4C | ||
+ | |hold | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | [[Image:HokkaidoU Japan 20100826a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]] | ||
+ | |||
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+ | *electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed. |
Revision as of 13:58, 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.
Reagent | Amount |
---|---|
colony solution | 10 uL |
DW | 23 uL |
10x PCR Buffer | 5 uL |
5 mM 4dNTPs | 5 uL |
25 mM MgSO4 | 3 uL |
EX-RBS Primer | 1.5 uL |
SlrP3 Primer | 1.5 uL |
KOD plus neo | 1 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
1-12K | 1 uL |
DW | 32.5 uL |
10x PCR Buffer | 5 uL |
5 mM 4dNTPs | 5 uL |
25 mM MgSO4 | 3 uL |
NLS Primer | 1.5 uL |
PS-R | 1.5 uL |
KOD plus neo | 1 uL |
Total | 50 uL |
- PCRed according to the table below.98C and 68C were 45 cycles.
temp | time |
---|---|
94C | 2 min |
98C | 10 sec |
68C | 60 sec |
4C | hold |
- electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed.