Team:HokkaidoU Japan/Notebook/September28
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*glycerol-stock of E.coli with salmonella's BAC library vector | *glycerol-stock of E.coli with salmonella's BAC library vector | ||
- | *making competent cell of E.coli with SPI2 | + | *making of competent cell of E.coli with SPI2 BAC vector |
*plasmid & GFP-double terminator's Ligation & Transformation | *plasmid & GFP-double terminator's Ligation & Transformation | ||
*PCR of E.coli with T3SSsignal and of GFP-double terminator | *PCR of E.coli with T3SSsignal and of GFP-double terminator | ||
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= PCR of Parts = | = PCR of Parts = | ||
- | + | We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be: | |
+ | <br> | ||
+ | EcoRI+XbaI+RBS+SlrP+SpeI+PstI | ||
+ | <br><br><br> | ||
+ | Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be: | ||
+ | <br><br> | ||
+ | EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI | ||
+ | <br><br> | ||
+ | PCR mix | ||
+ | <div style="float:left;"> | ||
{|style="text-align: center" class="protocol" | {|style="text-align: center" class="protocol" | ||
!Reagent | !Reagent | ||
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|1.5 uL | |1.5 uL | ||
|- | |- | ||
- | |SlrP3 | + | |SlrP3 Primer |
|1.5 uL | |1.5 uL | ||
|- | |- | ||
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|style="border-top:1px solid #996"|'''50 uL''' | |style="border-top:1px solid #996"|'''50 uL''' | ||
|} | |} | ||
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</div> | </div> | ||
<div style="float:left; margin-left:50px;"> | <div style="float:left; margin-left:50px;"> | ||
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{|style="text-align: center" class="protocol" | {|style="text-align: center" class="protocol" | ||
!Reagent | !Reagent | ||
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|style="border-top:1px solid #996"|'''50 uL''' | |style="border-top:1px solid #996"|'''50 uL''' | ||
|} | |} | ||
+ | </div> | ||
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+ | *PCRed according to the table below.98C and 68C for 45 cycles. | ||
+ | |||
+ | <br> | ||
+ | {|style="text-align: center" class="protocol" | ||
+ | !temp | ||
+ | !time | ||
+ | |- | ||
+ | |94C | ||
+ | |2 min | ||
+ | |- | ||
+ | |98C | ||
+ | |10 sec | ||
+ | |- | ||
+ | |68C | ||
+ | |60 sec | ||
+ | |- | ||
+ | |4C | ||
+ | |hold | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | *electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction. | ||
+ | Stored at -20C. |
Latest revision as of 17:32, 27 October 2010
- glycerol-stock of E.coli with salmonella's BAC library vector
- making of competent cell of E.coli with SPI2 BAC vector
- 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 which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:
EcoRI+XbaI+RBS+SlrP+SpeI+PstI
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI
PCR mix
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 for 45 cycles.
temp | time |
---|---|
94C | 2 min |
98C | 10 sec |
68C | 60 sec |
4C | hold |
- electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.