Team:HokkaidoU Japan/Notebook

From 2010.igem.org

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[[Team:HokkaidoU_Japan/Notebook|English]]
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[[Team:HokkaidoU_Japan/Notebook_jp|日本語]]
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==Calendar==
==Calendar==
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|style="color:red;"|[[#Sunday, October 3|3]]
|style="color:red;"|[[#Sunday, October 3|3]]
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|[[#Monday, October 4|4]]
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|[[Team:HokkaidoU_Japan/Notebook/October|4]]
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|[[#Tuesday, October 5|5]]
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|[[Team:HokkaidoU_Japan/Notebook/October|5]]
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|[[#Wednesday, October 6|6]]
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|[[Team:HokkaidoU_Japan/Notebook/October|6]]
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|[[#Thursday, October 7|7]]
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|[[Team:HokkaidoU_Japan/Notebook/October|7]]
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|[[#Friday, October 8|8]]
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|[[Team:HokkaidoU_Japan/Notebook/October|8]]
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|style="color:blue;"|[[#Saturday, October 9|9]]
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|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|9]]
|-
|-
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|style="color:red;"|[[#Sunday, October 10|10]]
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|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|10]]
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|[[#Monday, October 11|11]]
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|[[Team:HokkaidoU_Japan/Notebook/October|11]]
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|[[#Tuesday, October 12|12]]
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|[[Team:HokkaidoU_Japan/Notebook/October|12]]
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|[[#Wednesday, October 13|13]]
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|[[Team:HokkaidoU_Japan/Notebook/October|13]]
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|[[#Thursday, October 14|14]]
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|[[Team:HokkaidoU_Japan/Notebook/October|14]]
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|[[#Friday, October 15|15]]
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|[[Team:HokkaidoU_Japan/Notebook/October|15]]
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|style="color:blue;"|[[#Saturday, October 16|16]]
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|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|16]]
|-
|-
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|style="color:red;"|[[#Sunday, October 17|17]]
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|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|17]]
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|[[#Monday, October 18|18]]
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|[[Team:HokkaidoU_Japan/Notebook/October|18]]
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|[[#Tuesday, October 19|19]]
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|[[Team:HokkaidoU_Japan/Notebook/October|19]]
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|[[#Wednesday, October 20|20]]
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|[[Team:HokkaidoU_Japan/Notebook/October|20]]
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|[[#Thursday, October 21|21]]
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|[[Team:HokkaidoU_Japan/Notebook/October|21]]
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|[[#Friday, October 22|22]]
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|[[Team:HokkaidoU_Japan/Notebook/October|22]]
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|style="color:blue;"|[[#Saturday, October 23|23]]
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|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|23]]
|-
|-
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|style="color:red;"|[[#Sunday, October 24|24]]
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|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|24]]
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|[[#Monday, October 25|25]]
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|[[Team:HokkaidoU_Japan/Notebook/October|25]]
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|[[#Tuesday, October 26|26]]
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|[[Team:HokkaidoU_Japan/Notebook/October|26]]
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|[[#Wednesday, October 27|27]]
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|[[Team:HokkaidoU_Japan/Notebook/October|27]]
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|[[#Thursday, October 28|28]]
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|[[Team:HokkaidoU_Japan/Notebook/October|28]]
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|[[#Friday, October 29|29]]
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|[[Team:HokkaidoU_Japan/Notebook/October|29]]
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|style="color:blue;"|[[#Saturday, October 30|30]]
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|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|30]]
|-
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|style="color:red;"|[[#Sunday, October 31|31]]
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|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|31]]
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===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===
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=[[Team:HokkaidoU_Japan/Notebook/October|After October 3]]=
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===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===
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===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===
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===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===
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===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===
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===[[Team:HokkaidoU_Japan/Notebook/October9|Saturday, October 9]]===
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===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===
 
----
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===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===
 
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===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===
 

Latest revision as of 08:44, 21 April 2011

English / 日本語

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

Thursday, September 22

  • Digestion of pSB1A3, Arabinose promoter and GFP + double terminator
  • Ligation & Transformation

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

After October 3

Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook"