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Team:HokkaidoU Japan/Notebook/September28
From 2010.igem.org
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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 | ||
| Line 23: | Line 23: | ||
= 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;"> | <div style="float:left;"> | ||
{|style="text-align: center" class="protocol" | {|style="text-align: center" class="protocol" | ||
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| + | *PCRed according to the table below.98C and 68C for 45 cycles. | ||
| - | + | <br> | |
| - | + | ||
{|style="text-align: center" class="protocol" | {|style="text-align: center" class="protocol" | ||
!temp | !temp | ||
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|} | |} | ||
| - | *electrophoresed the two | + | *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
since 13 Jul 2010
since 1 Aug 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.
