Team:HokkaidoU Japan/Notebook/September28
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(Difference between revisions)
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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: | 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 | EcoRI+XbaI+RBS+SlrP+SpeI+PstI | ||
- | <br><br> | + | <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: | 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> | <br><br> |
Revision as of 15:37, 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
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 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.