Team:Chiba/jk/β

From 2008.igem.org

(Difference between revisions)
m (16 September, 2008)
(15 September, 2008)
Line 2,460: Line 2,460:
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:competent cells : BW⊿FliC
:competent cells : BW⊿FliC
 +
::*BBa_S03623 [http://partsregistry.org/Part:BBa_S03623]
::*BBa_K084007 [http://partsregistry.org/Part:BBa_K084007]
::*BBa_K084007 [http://partsregistry.org/Part:BBa_K084007]
::*BBa_K084008 [http://partsregistry.org/Part:BBa_K084008]
::*BBa_K084008 [http://partsregistry.org/Part:BBa_K084008]

Revision as of 04:39, 20 September 2008

β班の実験ノート

Contents

Week 1

17 August, 2008

Transformation
competent cells : XL10G
  • BBa_R0040(2008) [http://partsregistry.org/Part:BBa_R0040] -> No colonies on plates
  • BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] -> No colonies on plates
  • BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
  • BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
  • BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
  • BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] -> No colonies on plates

18 August, 2008

PCR
  • BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076]
  • BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077]
  • BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079]
  • BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase 1
Thermopol Buffer 10
dH2O 68
TOTAL 100μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃


--->Gel Check


Chiba-0818.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] ->None
  • BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] ->None
  • BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] ->None
  • BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] ->None
  • BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] ->None
  • BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control ->OK



Transformation

competent cells : XL10G

  • BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
  • BBa_C0076(2006) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates(19/8)
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]


--->(20/8)Mini prep

19 August, 2008

PCR
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154]
  • BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154]
  • BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase VENT 1
Thermopol Buffer 5
dH2O 28
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃


--->Gel Check
Chiba-0819.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] ->None
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] ->None
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] ->None
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] ->None
  • BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154] ->None
  • BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154] ->None
  • BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control ->OK


20 August, 2008

(18/8)--->Mini prep
  • BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]


--->Gel Check
Chiba-0820-3.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None


Chiba-0820-4.JPG
Sample DNA 10
Loading Dye 4
dH2O 10
TOTAL 24μL
From left;
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> OK
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
  • BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
  • BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK



Chiba-0820.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK


--->Digestion test

  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
Sample No 12345678
BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] 3---3---
BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -3---3--
BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] --3---3-
BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] ---3---3
EcoRⅠ 0.10.10.10.10.10.10.10.1
PstⅠ ----0.10.10.10.1
Buffer EcoRⅠ 11111111
BSA 11111111
dH2O 4.94.94.94.94.84.84.84.8
TOTAL 1010101010101010


--->Gel Check
Chiba-0820-2.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
(Single Digestion 1~4)
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
(Double Digestion 1~4)
  • BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
  • BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
  • BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK

21 August, 2008

(20/8)--->PCR
  • BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase TAQ 1
Thermopol Buffer 5
dH2O 28
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃


--->Gel Check
Chiba-0821.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
  • BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None
  • BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
  • BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
  • BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK


Transformation
competent cells : XL10G
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]



--->(23/8)Digestion


22 August, 2008

Transformation
competent cells : XL10G
  • BBa_J13002(2007) [http://partsregistry.org/Part:BBa_J13002]
  • BBa_J13002(2006) [http://partsregistry.org/Part:BBa_J13002]
  • BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
  • BBa_J04500(2006) [http://partsregistry.org/Part:BBa_J04500]
(Locate & 2 Punch ~iyama ver.~)
  • BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
  • BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
  • BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
  • BBa_J04500(2008) [http://partsregistry.org/Part:BBa_J04500] -> No colonies on plates


--->(24/8)Mini prep

23 August, 2008

(21/8)--->Mini prep
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]


--->Digestion test
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]


Sample No 1 2 3 4 5 6 7 8 9101112131415161718192021
BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] 3------3------3------
BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] -3------3------3-----
BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] --3------3------3----
BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] ---3------3------3---
BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] ----3------3------3--
BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] -----3------3------3-
BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] ------1------3------3
EcoRⅠ -------0.10.10.10.10.10.10.10.10.10.10.10.10.10.1
PstⅠ --------------0.10.10.10.10.10.10.1
Buffer EcoRⅠ -------0.90.90.90.90.90.90.90.80.80.80.80.80.80.8
BSA -------11111111111111
dH2O -------55555555555555
TOTAL 33333311010101010101010101010101010


--->Gel Check
Chiba-0823-1.JPG
Sample No 1~67
Sample DNA 31
Loading Dye 11
dH2O 24
TOTAL 66
From left; Sanple No. 1~7
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]



Chiba-0823-2.JPG
Sample No 8~21
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
(Single Digestion) Sanple No. 8~14
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] -> OK
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] -> OK
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] -> OK
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] -> OK
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] -> OK
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] -> OK
  • BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK
(Double Digestion) Sample No. 15~21
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] -> OK
  • BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] -> OK
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] -> OK
  • BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] -> OK
  • BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] -> OK
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] -> OK
  • BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK?



Digestion test
BBa_J04500(2007:old-sample)[http://partsregistry.org/Part:BBa_J04500]
Sample No 123
Sample DNA 333
EcoRⅠ -0.10.1
SpeⅠ --0.1
EcoRⅠ Buffer -0.90.8
BSA -11
dH2O -55
TOTAL 31010


--->Gel Check

Chiba-0823-3.JPG
Sample No 123
Sample DNA 31010
Loading Dye 122
dH2O 2--
TOTAL 61212
From left;
  1. BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK
  2. BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Single Digestion -> OK
  3. BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Double Digestion -> Bad

Week 2

24 August, 2008

(22/8)--->Mini prep
  1. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
  2. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
  3. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
  4. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)


--->Digestion test
  1. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
  2. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
  3. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
  4. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)


Sample No 5678
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) 1---
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -1--
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) --1-
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) ---1
EcoRⅠ 0.10.10.10.1
Buffer 4 0.90.90.90.9
dH2O 8888
TOTAL 10101010


--->Gel Check
Chiba-0824-1.JPG
Sample No 1~8
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  1. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> ?
  2. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
  3. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
  4. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
  5. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
  6. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
  7. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
  8. BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> None?
Chiba-0824-2.JPG
Sample No 5~8
Sample DNA 9
Loading Dye 2
dH2O 1
TOTAL 12

From the left; (single digestuion)

5, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
6, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
7, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
8, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK

25 August, 2008

(24/8)--->Digestion
  1. Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
  2. Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
  3. Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
  4. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Sample No 1234
Sample DNA 5511
EcoRⅠ 0.5o.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101
--->Gel Check
Chiba-0825-1.JPG
Sample No 1~34
Sample DNA 101
Loading Dye 21
dH2O -4
TOTAL 126
From right;
  1. Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
  2. Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
  3. Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
  4. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)


--->Digestion (Double Digestion)

  1. BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
  2. BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
  3. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
]

</tr>

Sample No 123
Sample DNA 33ng/μl×60μl33ng/μl×60μl100ng/μl×20μl
PstⅠ 222
XbaⅠ 22-
SpeⅠ --2
Buffer 2 --5
Buffer 3 1010-
BSA 10105
dH2O 161616
TOTAL 10010052

26 August, 2008

(8/25)--->Gel Check
Chiba-0825-2.JPG
Sample No 1~3
Sample DNA 30
Loading Dye 6
TOTAL 36
1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
Insert:609bp -> OK
Chiba-0825-3.JPG
2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
Insert:609bp -> OK
Chiba-0825-4.JPG
3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
-> OK


--->Gel Extract


--->Zymo Clean
  1. 5μl
  2. 5μl
  3. 27μl


--->Gel Check
Chiba-0825-5.JPG
Sample No 1~3
Sample DNA 2
Loading Dye 2
H2O 8
TOTAL 12
From left;
  1. insert:C0178 -> OK
  2. insert:C0170 -> OK?
  3. vector:J04500 -> OK


--->SAP
Sample No 3
Sample DNA 26
SAP 1
SAP Buffer 3
TOTAL 30μL


--->Zymo Clean
Sample No.3->9μl


--->Gel Check
Chiba-0826.JPG
Sample No.3 1
Loading Dye 1
dH2O 4
TOTAL 6
Sample No.3 -> OK ・・・30ng?


--->Ligation
]

</tr>

Sample No (1)(2)(3)(4)(5)
insert①C0178 1--1-
insert②C0170 -1--1
vector③J04500 --222
ligase 11111
Buffer 22222
dH2O 66544
TOTAL 1010101010


--->Transformation
  1. insert①C0178
  2. insert②C0170
  3. vector③J04500
  4. insert①C0178+vector③J04500
  5. insert②C0170+vector③J04500


27 August, 2008

Digestion Test

Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]
Sample No 123
Sample DNA 111
PstⅠ --0.5
EcoRⅠ -0.50.5
Buffer 3 -11
BSA --1
dH2O -7.52
TOTAL 11010


--->Gel Check
Chiba-0827.JPG
Sample No 123
Sample DNA 155
Loading Dye 111
H2O 4--
TOTAL 666
From left;
  1. BBa_R0010
  2. Single Digestion
  3. Double Digestion


(26/8)--->Colony-PCR
Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 5
Reverse Primer 5
DNA polymerase 0.5
Thermopol Buffer 5
dH2O 28.5
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃



--->Gel Check
Chiba-0827-2.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
1~5 insert:C0170 + vector:J04500 -> OK
6 F2620・・Positive Control -> OK
7~11 insert:C0178 + vector:J04500 -> OK


--->(28/8)Miniprep


Transformation

competent cells : XL10G
  • BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  • BBa_C0062(2006) [http://partsregistry.org/Part:BBa_C0062] -> no colony on plates
  • BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
  • BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
  • BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  • BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]


--->Mini prep


--->(29/8)Gel Check


28 August, 2008

(27/8)--->Mini prep
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500
Sample No 3456
insert:C0170 + vector:J04500 1-1-
insert:C0178 + vector:J04500 -1-1
EcoRⅠ 0.10.10.10.1
SpeⅠ --0.10.1
Buffer 3 1111
BSA --11
dH2O 7.87.86.86.8
TOTAL 10101010


--->(29/8)Gel Check
Chiba-0828.JPG
Sample No 1,23~6
Sample DNA 110
Loading Dye 12
H2O 4-
TOTAL 612
From left;
  1.  -> OK
  2.  -> OK
  3.  -> OK
  4.  -> OK
  5.  -> OK
  6.  -> OK

29 August, 2008

--->Digestion Test
  1. BBa_R0010(2007) [http://partsregistry.org/Part:BBa_R0010]
  2. BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  3. BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
  4. BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
  5. BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  6. BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
  7. BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  8. BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
  9. BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  10. BBa_S03160(2006) [http://partsregistry.org/Part:BBa_S03160]


Sample No S1~10W1W2~10
Sample DNA 142
PstⅠ -0.0750.1
EcoRⅠ 0.10.0750.1
Buffer 3 10.751
BSA -0.751
dH2O 7.94.355.8
TOTAL 101010


--->Gel Check
Chiba-0829.JPG
Sample No. 1~10S1~S10,W1~W10
Sample DNA 110
Loading Dye 12
TOTAL 612
From left;
1~5,S1,S5,W1~W5
Chiba-0829-2.JPG
From left;
6~10,S6~S10,W6~W10


(27/8)--->Gel Check
Chiba-0829-3.JPG
Sample DNA 0.5
Loading Dye 1
dH2O 4.5
TOTAL 6μL
R0010 -> OK


Digestion

  1. BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  2. BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  3. BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  4. BBa_R0010(2006) [http://partsregistry.org/Part:BBa_R0010]


Sample No 1~34
Sample DNA 100ng/μL×20200ng/μL×30
PstⅠ 22
XbaⅠ 2-
SpeⅠ -2
Buffer 2 -5
Buffer 3 5-
BSA 55
dH2O 166
TOTAL 5050
--->Gel Check
Chiba-0829-4.JPG
Sample No. 1~34
Sample DNA 103
Loading Dye 22
dH2O -7
TOTAL 1212
insert-I9026 : 685bp -> None
Chiba-0829-5.JPG
insert-I9030 : 745bp -> None
Chiba-0829-6.JPG
insert-S03154 : 685bp -> None
Chiba-0829-7.JPG
vector-R0010 : 2079bp -> OK


--->Gel Extract


--->Zymo Clean
->15μL
--->SAP
Sample No 4
Sample DNA 15
SAP 2
SAP Buffer 3
dH2O 10
TOTAL 30μL


--->Zymo Clean
--->Suspension! (We got information, by the team-output, to the effect that the density of the sample is too low.)


30 August, 2008

Transformation
competent cells : XL10G
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  • BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  • BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
--->(31/8)Mini prep


(29/8)--->Mini prep
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  • insert:C0170 + vector:J04500 -> Sample No.1~4
  • insert:C0178 + vector:J04500 -> Sample No.5~8
  • BBa_T9002(2007)[http://partsregistry.org/Part:BBa_T9002] -> Sample No.9
  • Single Digestion of Sample No.1~9 -> Sample No.10~18
  • Double Digestion of Sample No.1~9 -> Sample No.19~27
Sample No.Single : 10~18Double : 19~27
Sample DNA15
XbaⅠ0.10.1
SpeⅠ-0.1
Buffer 211
BSA11
dH2O6.92.8
TOTAL1010


--->Gel Check
Chiba-0830.JPG
Sample No 1~910~1819~27
Sample DNA 11010
Loading Dye 122
dH2O 4--
TOTAL 61212
From left;
Sanple No.1~13
-> OK
Chiba-0830-2.JPG
From left;
Sample No.14~17,24~16
14 -> OK
15,16 -> Bad
17 -> None
24~16 -> Bad
Chiba-0830-3.JPG
From left;
Sample No.18,27,19~23
18,27 -> Bad
19~22 -> OK
23 -> OK??

Week 3

31 August, 2008

Transformation
Competent Cells : XL10G
  • Plac+RBS+LasI
  • Plac+RBS+RhlI

Digestion

  1. BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
  2. BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
  3. BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
  4. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Sample No 1~34
Sample DNA1210
PstⅠ 0.20.2
XbaⅠ0.2-
SpeⅠ-0.4
Buffer 2 -1.5
Buffer 3 2-
BSA 21.5
dH2O 3.61.4
TOTAL 2015


--->(1/9)Gel Check


(30/8)--->Mini prep


--->Digestion test
  • Plac+RBS+RhlI Sample No.2~5
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
Sample Single Digesiton 2~5Double Digestion 2~5Single Digestion T9002①Single Digesiton T9002②<Double Digestion T9002①②
Sample DNA13313
XbaⅠ0.10.10.10.10.1
SpeⅠ-0.1--0.1
Buffer 2 0.90.80.90.90.8
BSA 11111
dH2O 75575
TOTAL 1010101010


--->Gel Check

Chiba-0831-p87.JPG
Sample No Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①② Single & Double Digestion
Sample DNA 110
Loading Dye 12
dH2O 4-
TOTAL 612
From Left
  • Single Digestion Plac+RBS+RhlI 2~5
  • Single Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②

1 September, 2008

(31/8)--->Gel Check
Chiba-0901.JPG
Sample No 1~34
Sample DNA 2015
Loading Dye 43
TOTAL 2418
From left;
insert-1(I9026)
insert-2(I9030)
Chiba-0901-2.JPG
insert-3(S03154)
Chiba-0901-3.JPG
vector-4(R0010)
--->zymo
vector-4(R0010)
-> 15μL
--->SAP
vectr-4(R0010)
--->Zymo
1 -> 7μL
2 -> 7
3 -> 7
4 -> 20


Transformation

Competent cells : BW ΔFliC 40μL
  • Plac+RBS+LasI : Sample No.L1~L4
  • Plac+RBS+RhlI : Sample No.R1~R4
--->(2/9)OD

2 September, 2008

(31/8)--->Gel Check
Chiba-0902.JPG
Sample No
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
I9026
I9030
S03154


(1/9)--->Colony-PCR
Colony PCR of 8 colonies from ligation plates (1/9-(1),(2)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 0.3
Reverse Primer 0.3
DNA polymerase TAQ 0.5
Thermopol Buffer 3
dH2O 20.5
TOTAL 30μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃


--->Gel Check

Chiba-0902-2.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • Plac+RBS+RhlI+LVA
R1 -> OK
R2 -> Bad
R3~R7 -> OK
R8 -> Bad
Chiba-0902-3.JPG
From left;
  • Plac+RBS+CinI+LVA
C1,C2 -> OK
C3 -> Bad
C4~C6 -> OK
Chiba-0902-4.JPG
From left;
  • Plac+RBS+CinI+LVA
C7,C8 -> OK
  • BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->OD測


Transformation

Competent cells : XL10G 30μL
  • C0161(2007)
  • C0161(2006)
  • C0261(2007)
  • C0261(2006)
--->(4/9)miniprep

3 September, 2008

(2/9)--->Mini prep
--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • S03154 -> OK
  • R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK
Chiba-0903-2.JPG
From left;
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK
--->Digestion test
  • R1,R3~R7 (Plac+RBS+RhlI+LVA)
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA)
Digestion SingleDouble
Sample DNA 13
XbaⅠ 0.10.1
PstⅠ 0.10.1
Buffer 2 0.9-
Buffer 3 --0.8
BSA 11
dH2O 75
TOTAL 1010


--->Gel Check
Chiba-0903-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
  • Single Digestion : R1,R3~R7 -> OK
  • Single Digestion : C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : R1,R3~R7 -> OK
  • Double Digestion : C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test : mix

Sample No. 1234567891011
L1 2----------
L2-2---------
L3--2--------
L4---2-------
R1----1------
R2-----1-----
R3------1----
R4-------1---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------2--
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]22221111211
AHL11111111111
|
| 8h
V
Spindown
|
|
V
the intensity GFP

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample No
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-2.JPG
Sample No
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-3.JPG
Sample No
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;

5 September, 2008

6 September, 2008

Week 4

7 September, 2008

8 September, 2008

9 September, 2008

10 September, 2008

11 September, 2008

12 September, 2008

13 September, 2008

Gel Check
  1. BBa_C0161[http://partsregistry.org/Part:BBa_Co161](2007)
  2. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
  3. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Chiba-0913.JPG
Sample No 1~3
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;


--->[Team:Chiba/protocol/digestion|Digestion]]

  1. BBa_C0161[http://partsregistry.org/Part:BBa_Co161](2007)
  2. BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
  3. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)


Sample No
Sample DNA 5511
EcoRⅠ 0.5o.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101

Week 5

14 September, 2008

(13/9)--->Mini prep


Transformation
Competent cells : XL10G
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002](2007)
  • Plac+RhlI+LVA
  • Plac+CinI+LVA
--->(15/9)

15 September, 2008

Transformation
competent cells : BW⊿FliC
  • BBa_S03623 [http://partsregistry.org/Part:BBa_S03623]
  • BBa_K084007 [http://partsregistry.org/Part:BBa_K084007]
  • BBa_K084008 [http://partsregistry.org/Part:BBa_K084008]
  • BBa_K084009 [http://partsregistry.org/Part:BBa_K084009]
  • BBa_T9002 [http://partsregistry.org/Part:BBa_T9002]

cultured (15/9)pre-cultures in LB medium.
added AHL(total concentration was 0nM,1nM,10nM,100nM,1000nM)

-->check green fluorescence intensity(Excitation:485nm,Emission:527nm)


pre-Cultured transformants(LB,37℃,14h)

  • BBa_K084007~BBa_K084009
  • BBa_T9002
  • BBa_S03623

cultured above pre-cultures
-->mix(16/9)

16 September, 2008

mix:(15/9)cultures in the way prescribed in Table 1 below.

-->check green fluorescence intensity(Excitation:485nm,Emission:527nm)


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