Team:HokkaidoU Japan/Notebook

From 2010.igem.org

(Difference between revisions)
(Replacing page with '{{Template:HokkaidoU_Japan}} =Notebook= Coming soon')
 
(7 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
-
=Notebook=
+
__NOTOC__
-
Coming soon
+
<html>
 +
<style>
 +
#main h3{
 +
  font-size: 116%;
 +
  font-weight:normal;
 +
  text-decoration:none;
 +
}
 +
 
 +
#main h3 a{
 +
  font-size: 116%;
 +
  font-weight:normal;
 +
  text-decoration:none;
 +
}
 +
 
 +
#main ul {
 +
  font-family:cursive;
 +
}
 +
 
 +
 
 +
.calendar {
 +
  background-color:#F1F0E7;
 +
  text-align:center;
 +
  border:1px solid #630;
 +
  float:left;
 +
  margin:0px 10px 0px 5px;
 +
  width:180px;
 +
}
 +
 
 +
.calendar a {
 +
  display: block;
 +
  font-weight: bold;
 +
  text-decoration: none;
 +
}
 +
 
 +
</style>
 +
</html>
 +
 
 +
[[Team:HokkaidoU_Japan/Notebook|English]]
 +
/
 +
[[Team:HokkaidoU_Japan/Notebook_jp|日本語]]
 +
----
 +
 
 +
==Calendar==
 +
{|class="calendar"
 +
|-
 +
|colspan="7"|July
 +
|-
 +
!style="color:red;"|S
 +
!M
 +
!T
 +
!W
 +
!T
 +
!F
 +
!style="color:blue;"|S
 +
|-
 +
|style="color:red;"|
 +
|
 +
|
 +
|
 +
|1
 +
|2
 +
|style="color:blue;"|3
 +
|-
 +
|style="color:red;"|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|style="color:blue;"|10
 +
|-
 +
|style="color:red;"|11
 +
|[[#Monday, July 12|12]]
 +
|13
 +
|14
 +
|15
 +
|16
 +
|style="color:blue;"|17
 +
|-
 +
|style="color:red;"|18
 +
|19
 +
|20
 +
|[[#Wednesday, July 21|21]]
 +
|22
 +
|23
 +
|style="color:blue;"|24
 +
|-
 +
|style="color:red;"|25
 +
|[[#Monday, July 26|26]]
 +
|[[#Tuesday, July 27|27]]
 +
|28
 +
|29
 +
|30
 +
|style="color:blue;"|31
 +
|-
 +
|&nbsp;
 +
|}
 +
 
 +
{|class="calendar"
 +
|-
 +
|colspan="7"|August
 +
|-
 +
!style="color:red;"|S
 +
!M
 +
!T
 +
!W
 +
!T
 +
!F
 +
!style="color:blue;"|S
 +
|-
 +
|style="color:red;"|1
 +
|[[#Monday, August 2|2]]
 +
|3
 +
|4
 +
|5
 +
|6
 +
|style="color:blue;"|7
 +
|-
 +
|style="color:red;"|8
 +
|[[#Monday, August 9|9]]
 +
|[[#Tuesday, August 10|10]]
 +
|[[#Wednesday, August 11|11]]
 +
|[[#Thursday, August 12|12]]
 +
|[[#Friday, August 13|13]]
 +
|style="color:blue;"|14
 +
|-
 +
|style="color:red;"|15
 +
|[[#Monday, August 16|16]]
 +
|[[#Tuesday, August 17|17]]
 +
|[[#Wednesday, August 18|18]]
 +
|[[#Thursday, August 19|19]]
 +
|[[#Friday, August 20|20]]
 +
|style="color:blue;"|21
 +
|-
 +
|style="color:red;"|22
 +
|[[#Monday, August 23|23]]
 +
|[[#Tuesday, August 24|24]]
 +
|[[#Wednesday, August 25|25]]
 +
|[[#Thursday, August 26|26]]
 +
|[[#Friday, August 27|27]]
 +
|style="color:blue;"|28
 +
|-
 +
|style="color:red;"|29
 +
|[[#Monday, August 30|30]]
 +
|[[#Tuesday, August 31|31]]
 +
|
 +
|
 +
|
 +
|style="color:blue;"|
 +
|-
 +
|&nbsp;
 +
|}
 +
 
 +
{|class="calendar"
 +
|-
 +
|colspan="7"|September
 +
|-
 +
!style="color:red;"|S
 +
!M
 +
!T
 +
!W
 +
!T
 +
!F
 +
!style="color:blue;"|S
 +
|-
 +
|style="color:red;"|
 +
|
 +
|
 +
|[[#Wednesday, September 1|1]]
 +
|[[#Thursday, September 2|2]]
 +
|[[#Friday, September 3|3]]
 +
|style="color:blue;"|[[#Saturday, September 4|4]]
 +
|-
 +
|style="color:red;"|5
 +
|[[#Monday, September 6|6]]
 +
|[[#Tuesday, September 7|7]]
 +
|[[#Wednesday, September 8|8]]
 +
|[[#Thursday, September 9|9]]
 +
|[[#Friday, September 10|10]]
 +
|style="color:blue;"|11
 +
|-
 +
|style="color:red;"|12
 +
|[[#Monday, September 13|13]]
 +
|[[#Tuesday, September 14|14]]
 +
|[[#Wednesday, September 15|15]]
 +
|[[#Thursday, September 16|16]]
 +
|[[#Friday, September 17|17]]
 +
|style="color:blue;"|18
 +
|-
 +
|style="color:red;"|19
 +
|20
 +
|[[#Tuesday, September 21|21]]
 +
|[[#Wednesday, September 22|22]]
 +
|[[#Thursday, September 23|23]]
 +
|[[#Friday, September 24|24]]
 +
|style="color:blue;"|25
 +
|-
 +
|style="color:red;"|26
 +
|[[#Monday, September 27|27]]
 +
|[[#Tuesday, September 28|28]]
 +
|[[#Wednesday, September 29|29]]
 +
|[[#Thursday, September 30|30]]
 +
|
 +
|
 +
|-
 +
|&nbsp;
 +
|}
 +
 
 +
{|class="calendar"
 +
|-
 +
|colspan="7"|October
 +
|-
 +
!style="color:red;"|S
 +
!M
 +
!T
 +
!W
 +
!T
 +
!F
 +
!style="color:blue;"|S
 +
|-
 +
|style="color:red;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|1
 +
|style="color:blue;"|[[#Saturday, October 2|2]]
 +
|-
 +
|style="color:red;"|[[#Sunday, October 3|3]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|4]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|5]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|6]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|7]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|8]]
 +
|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|9]]
 +
|-
 +
|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|10]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|11]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|12]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|13]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|14]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|15]]
 +
|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|16]]
 +
|-
 +
|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|17]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|18]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|19]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|20]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|21]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|22]]
 +
|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|23]]
 +
|-
 +
|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|24]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|25]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|26]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|27]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|28]]
 +
|[[Team:HokkaidoU_Japan/Notebook/October|29]]
 +
|style="color:blue;"|[[Team:HokkaidoU_Japan/Notebook/October|30]]
 +
|-
 +
|style="color:red;"|[[Team:HokkaidoU_Japan/Notebook/October|31]]
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:#555;"|
 +
|style="color:blue;"|
 +
|}
 +
<div style="clear:both"></div>
 +
 
 +
==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)
 +
----
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August2|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
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August10|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
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===
 +
* Preparation for the next day's miniprep
 +
* Estimation of enzyme activity
 +
* Preparation of the glycerol stocks
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===
 +
* Miniprep (1-18F, 2-21H and 2-11P)
 +
* Electrophoresis of the DNA which had been amplified via PCR and miniprep
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===
 +
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)
 +
----
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August16|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)
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===
 +
* Purification of the DNA solutions via gel extraction
 +
* Preparation of agar medium containing 35 ug/mL chloramphenicol
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===
 +
* Digestion and gel extraction of 1-2M (retry)
 +
* 3 piece ligation of 1-18F, 1-23L and pSB1C3
 +
* Transformation
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August19|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
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===
 +
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3
 +
----
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===
 +
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)
 +
* PCR amplification of BioBrick parts using new primers
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August24|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
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August25|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
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August26|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)
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August27|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
 +
----
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===
 +
* Measurement of restriction enzyme activity using highly purified pUC119
 +
* Digestion of PCR products that had been amplified using new primers
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===
 +
* Gel extraction, ligation and transformation of pUC119
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===
 +
* PCR amplification of 1-5A (RFP reporter)
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===
 +
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===
 +
* Colony PCR of ''E. coli''  transformed on previous day
 +
* PCR amplification of 1-3A (RFP reporter)
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===
 +
* PCR amplification of 1-5A (RFP reporter)
 +
* Estimation of the amount of 1-3A PCR product
 +
 
 +
----
 +
 
 +
===[[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]]===
 +
*Concentration check of DNA used for ligation yesterday
 +
*Ethanol precipitation
 +
*Colony PCR of competent cells
 +
 
 +
===[[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]]===
 +
* 3 piece ligation of HSP, GFP and pSB1C3
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===
 +
*3 piece ligation, continued
 +
----
 +
 
 +
===[[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]]===
 +
* Digestion and ligation of pSB1C3, araC Promoter and GFP
 +
* Ethanol precipitation
 +
 
 +
===[[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]]===
 +
*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]]===
 +
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September22|Thursday, September 22]]===
 +
*Digestion of pSB1A3, Arabinose promoter and GFP + double terminator
 +
*Ligation & Transformation
 +
 
 +
===[[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]]===
 +
*Miniprep of Arac+RBS+pSB1A3
 +
*Follow quality check
 +
----
 +
 
 +
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===
 +
* Culture of the BAC clones
 +
 
 +
===[[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]]===
 +
*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]]===
 +
*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/October|After October 3]]=
 +
 
 +
----

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"