Team:HokkaidoU Japan/NotebookTest
From 2010.igem.org
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+ | ==Calendar== | ||
{|class="calendar" | {|class="calendar" | ||
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|colspan="7"|July | |colspan="7"|July | ||
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|colspan="7"|August | |colspan="7"|August | ||
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|colspan="7"|September | |colspan="7"|September | ||
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|[[#Tuesday, September 21|21]] | |[[#Tuesday, September 21|21]] | ||
|[[#Wednesday, September 22|22]] | |[[#Wednesday, September 22|22]] | ||
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|colspan="7"|October | |colspan="7"|October | ||
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|1 | |1 | ||
- | |style="color:blue;"|2 | + | |style="color:blue;"|[[#Saturday, October 2|2]] |
|- | |- | ||
- | |style="color:red;"|3 | + | |style="color:red;"|[[#Sunday, October 3|3]] |
- | |4 | + | |[[#Monday, October 4|4]] |
- | |5 | + | |[[#Tuesday, October 5|5]] |
- | |6 | + | |[[#Wednesday, October 6|6]] |
- | |7 | + | |[[#Thursday, October 7|7]] |
- | |8 | + | |[[#Friday, October 8|8]] |
- | |style="color:blue;"|9 | + | |style="color:blue;"|[[#Saturday, October 9|9]] |
|- | |- | ||
- | |style="color:red;"|10 | + | |style="color:red;"|[[#Sunday, October 10|10]] |
- | |11 | + | |[[#Monday, October 11|11]] |
- | |12 | + | |[[#Tuesday, October 12|12]] |
- | |13 | + | |[[#Wednesday, October 13|13]] |
- | |14 | + | |[[#Thursday, October 14|14]] |
- | |15 | + | |[[#Friday, October 15|15]] |
- | |style="color:blue;"|16 | + | |style="color:blue;"|[[#Saturday, October 16|16]] |
|- | |- | ||
- | |style="color:red;"|17 | + | |style="color:red;"|[[#Sunday, October 17|17]] |
- | |18 | + | |[[#Monday, October 18|18]] |
- | |19 | + | |[[#Tuesday, October 19|19]] |
- | |20 | + | |[[#Wednesday, October 20|20]] |
- | |21 | + | |[[#Thursday, October 21|21]] |
- | |22 | + | |[[#Friday, October 22|22]] |
- | |style="color:blue;"|23 | + | |style="color:blue;"|[[#Saturday, October 23|23]] |
|- | |- | ||
- | |style="color:red;"|24 | + | |style="color:red;"|[[#Sunday, October 24|24]] |
- | |25 | + | |[[#Monday, October 25|25]] |
- | |26 | + | |[[#Tuesday, October 26|26]] |
- | |27 | + | |[[#Wednesday, October 27|27]] |
- | |28 | + | |[[#Thursday, October 28|28]] |
- | |29 | + | |[[#Friday, October 29|29]] |
- | |style="color:blue;"|30 | + | |style="color:blue;"|[[#Saturday, October 30|30]] |
|- | |- | ||
- | |style="color:red;"|31 | + | |style="color:red;"|[[#Sunday, October 31|31]] |
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<div style="clear:both"></div> | <div style="clear:both"></div> | ||
- | =Abstract= | + | |
- | === | + | ==Abstract== |
+ | ===Monday, July 12=== | ||
* Our first experiment, transformation of BioBrick devices. | * Our first experiment, transformation of BioBrick devices. | ||
---- | ---- | ||
- | === | + | ===Wednesday, July 21=== |
* Preparation of competent cells | * Preparation of competent cells | ||
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12 | * Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12 | ||
- | === | + | ===Monday, July 26=== |
* Cultivation of transformed ''E. coli'' for the next day's miniprep | * Cultivation of transformed ''E. coli'' for the next day's miniprep | ||
- | === | + | ===Tuesday, July 27=== |
- | * Miniprep | + | * Miniprep (Alkaline SDS method) |
---- | ---- | ||
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===Friday, August 6=== | ===Friday, August 6=== | ||
---- | ---- | ||
- | === | + | ===Monday, August 9=== |
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli | * Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli | ||
* These are preliminary experiments before starting the main project | * These are preliminary experiments before starting the main project | ||
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* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium | * Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium | ||
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation | * Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation | ||
- | |||
- | |||
* Digestion of PCR products that had been amplified by using new primers | * Digestion of PCR products that had been amplified by using new primers | ||
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===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]=== | ||
+ | *Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter) | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]=== | ||
+ | *Concentration check of DNA used for ligation yesterday | ||
+ | *Ethanol precipitation | ||
+ | *Colony PCR of competent cells | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]=== | ||
+ | *Colony PCR | ||
+ | *Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3 | ||
+ | *PCR of GFP | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]=== | ||
+ | * 3 piece ligation of HSP, GFP and pSB1C3 | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]=== | ||
+ | *3 piece ligation, continued | ||
---- | ---- | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]=== | ||
+ | * AraC promoter purification | ||
+ | * And follow up checks for quality | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]=== | ||
+ | * Digestion and ligation of pSB1C3, araC Promoter and GFP | ||
+ | * Ethanol precipitation | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]=== | ||
- | === | + | *Observed results of yesterdays transformation |
+ | **Transformation using heat shock went well | ||
+ | **Electroporation transformation failed produce colonies | ||
+ | *Did Colony PCR of yesterdays transformed colonies | ||
+ | *Introduced colonies to L(+)Arabinose medium to check if it would show desired function | ||
+ | **Check for results tomorrow | ||
+ | |||
+ | ===Thursday, September 16=== | ||
+ | *Observed results of overnight incubation | ||
+ | **Fluorescence was visible when viewed by fluorescence microscope | ||
+ | *Did experiment to see if fluorescence is affected by arabinose concentration | ||
+ | *Scanned GFP intensity of broth containing colonies we isolated yesterday | ||
+ | *Had a free time so amplified some parts for easy training constructs | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]=== | ||
+ | *Construction of GFP marker for a part which will be secreted using T3SS | ||
+ | *Ordered primers for construction for same part | ||
---- | ---- | ||
- | === | + | |
+ | ===Monday, September 20=== | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]=== | ||
- | + | *Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos | |
+ | |||
+ | ===Wednesday, September 22=== | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]=== | ||
+ | *Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification | ||
+ | *Colony PCR of AraC+RBS+pSB1A3 | ||
+ | *Electroporetion of BAC plasmid into DH5α MG1655 | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]=== | ||
+ | *Miniprep of Arac+RBS+pSB1A3 | ||
+ | *Follow quality check | ||
---- | ---- | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]=== | ||
+ | * Culture of the BAC clones | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]=== | ||
+ | *glycerol-stock of E.coli with salmonella's BAC library vector | ||
+ | *plasmid & GFP-double terminator's Ligation & Transformation | ||
+ | *PCR of E.coli with T3SSsignal and of GFP-double terminator | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]=== | ||
+ | *Electrophoresis of T3SS signal and GFP + double terminator | ||
+ | *Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator | ||
+ | *making competent cell of E.coli with SPI2 | ||
+ | *PCR of T3SS signal Again | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]=== | ===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]=== | ||
- | ===[[Team:HokkaidoU_Japan/Notebook/ | + | *Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3 |
+ | *Transformation | ||
+ | |||
+ | ===Friday, October 1=== | ||
+ | |||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]=== | ||
+ | * Colony PCR | ||
+ | * Preparation for Sequencing | ||
+ | |||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]=== | ||
+ | * Miniprep | ||
---- | ---- | ||
+ | |||
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]=== | ||
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]=== | ||
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===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]=== | ||
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]=== | ||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October9|Saturday, October 9]]=== | ||
+ | |||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]=== | ||
---- | ---- | ||
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]=== | ||
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===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]=== | ||
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]=== | ||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]=== | ||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]=== | ||
---- | ---- | ||
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]=== | ||
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===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]=== | ||
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]=== | ||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]=== | ||
+ | ===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]=== | ||
---- | ---- | ||
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]=== | ||
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===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]=== | ||
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]=== | ===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]=== | ||
- | + | ===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]=== | |
- | ===[[Team:HokkaidoU_Japan/Notebook/ | + | ===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]=== |
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Latest revision as of 17:53, 27 October 2010
Calendar
July | ||||||
S | M | T | W | T | F | S |
---|---|---|---|---|---|---|
1 | 2 | 3 | ||||
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
August | ||||||
S | M | T | W | T | F | S |
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | ||||
September | ||||||
S | M | T | W | T | F | S |
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | |||
5 | 6 | 7 | 8 | 9 | 10 | 11 |
12 | 13 | 14 | 15 | 16 | 17 | 18 |
19 | 20 | 21 | 22 | 23 | 24 | 25 |
26 | 27 | 28 | 29 | 30 | ||
October | ||||||
S | M | T | W | T | F | S |
---|---|---|---|---|---|---|
1 | 2 | |||||
3 | 4 | 5 | 6 | 7 | 8 | 9 |
10 | 11 | 12 | 13 | 14 | 15 | 16 |
17 | 18 | 19 | 20 | 21 | 22 | 23 |
24 | 25 | 26 | 27 | 28 | 29 | 30 |
31 |
Abstract
Monday, July 12
- Our first experiment, transformation of BioBrick devices.
Wednesday, July 21
- Preparation of competent cells
- Single colony isolation of E. coli which had been transformed on Monday, July 12
Monday, July 26
- Cultivation of transformed E. coli for the next day's miniprep
Tuesday, July 27
- Miniprep (Alkaline SDS method)
Monday, August 2
- Restriction enzyme digestion of plasmids which had been purified by miniprep
- Electrophoresis assay
Tuesday, August 3
Wednesday, August 4
Thursday, August 5
Friday, August 6
Monday, August 9
- Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive E. coli and heat-sensitive E. coli
- These are preliminary experiments before starting the main project
Tuesday, August 10
- PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm
Wednesday, August 11
- Preparation for the next day's miniprep
- Estimation of enzyme activity
- Preparation of the glycerol stocks
Thursday, August 12
- Miniprep (1-18F, 2-21H and 2-11P)
- Electrophoresis of the DNA which had been amplified via PCR and miniprep
Friday, August 13
- PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)
Monday, August 16
- Measurement of restriction enzyme activity
- Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)
Tuesday, August 17
- Purification of the DNA solutions via gel extraction
- Preparation of agar medium containing 35 ug/mL chloramphenicol
Wednesday, August 18
- Digestion and gel extraction of 1-2M (retry)
- 3 piece ligation of 1-18F, 1-23L and pSB1C3
- Transformation
Thursday, August 19
- 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)
- Ligation that uses only vector pSB1C3 to estimate its efficiency
Friday, August 20
- PCR amplification of pSB1A3, pSB1C3 and pSB1T3
Monday, August 23
- PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)
- PCR amplification of BioBrick parts using new primers
Tuesday, August 24
- Electrophoresis of PCR products that had been amplified using new primers
- Examination of two ligation kits
- Electrophoresis assay of miscellaneous DNA solutions
- Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium
Wednesday, August 25
- Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium
- Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation
- Digestion of PCR products that had been amplified by using new primers
Thursday, August 26
- Electriphoresis to check whether ligation had been successful
- Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)
Friday, August 27
- Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation
- Retry of 3 piece ligation which was done on August 19th and 20th
- Ligation of vectors to each other
Monday, August 30
- Measurement of restriction enzyme activity using highly purified pUC119
- Digestion of PCR products that had been amplified using new primers
Tuesday, August 31
- Gel extraction, ligation and transformation of pUC119
Wednesday, September 1
- PCR amplification of 1-5A (RFP reporter)
Thursday, September 2
- Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119
Friday, September 3
- Colony PCR of E. coli transformed on previous day
- PCR amplification of 1-3A (RFP reporter)
Saturday, September 4
- PCR amplification of 1-5A (RFP reporter)
- Estimation of the amount of 1-3A PCR product
Monday, September 6
- Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)
Tuesday, September 7
- Concentration check of DNA used for ligation yesterday
- Ethanol precipitation
- Colony PCR of competent cells
Wednesday, September 8
- Colony PCR
- Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3
- PCR of GFP
Thursday, September 9
- 3 piece ligation of HSP, GFP and pSB1C3
Friday, September 10
- 3 piece ligation, continued
Monday, September 13
- AraC promoter purification
- And follow up checks for quality
Tuesday, September 14
- Digestion and ligation of pSB1C3, araC Promoter and GFP
- Ethanol precipitation
Wednesday, September 15
- Observed results of yesterdays transformation
- Transformation using heat shock went well
- Electroporation transformation failed produce colonies
- Did Colony PCR of yesterdays transformed colonies
- Introduced colonies to L(+)Arabinose medium to check if it would show desired function
- Check for results tomorrow
Thursday, September 16
- Observed results of overnight incubation
- Fluorescence was visible when viewed by fluorescence microscope
- Did experiment to see if fluorescence is affected by arabinose concentration
- Scanned GFP intensity of broth containing colonies we isolated yesterday
- Had a free time so amplified some parts for easy training constructs
Friday, September 17
- Construction of GFP marker for a part which will be secreted using T3SS
- Ordered primers for construction for same part
Monday, September 20
Tuesday, September 21
- Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos
Wednesday, September 22
Thursday, September 23
- Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
- Colony PCR of AraC+RBS+pSB1A3
- Electroporetion of BAC plasmid into DH5α MG1655
Friday, September 24
- Miniprep of Arac+RBS+pSB1A3
- Follow quality check
Monday, September 27
- Culture of the BAC clones
Tuesday, September 28
- glycerol-stock of E.coli with salmonella's BAC library vector
- plasmid & GFP-double terminator's Ligation & Transformation
- PCR of E.coli with T3SSsignal and of GFP-double terminator
Wednesday, September 29
- Electrophoresis of T3SS signal and GFP + double terminator
- Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator
- making competent cell of E.coli with SPI2
- PCR of T3SS signal Again
Thursday, September 30
- Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
- Transformation
Friday, October 1
Saturday, October 2
- Colony PCR
- Preparation for Sequencing
Sunday, October 3
- Miniprep