Team:Chiba/jk/β

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>実験ログ

β班の実験ノート
weeks 1-2
weeks 3-4

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

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

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

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

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

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	1	2	3	4	5	6	7	8
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	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
PstⅠ	-	-	-	-	0.1	0.1	0.1	0.1
Buffer EcoRⅠ	1	1	1	1	1	1	1	1
BSA	1	1	1	1	1	1	1	1
dH2O	4.9	4.9	4.9	4.9	4.8	4.8	4.8	4.8
TOTAL	10	10	10	10	10	10	10	10

--->Gel Check

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

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	9	10	11	12	13	14	15	16	17	18	19	20	21
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	-	-	-	-	-	-	-	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
PstⅠ	-	-	-	-	-	-	-	-	-	-	-	-	-	-	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Buffer EcoRⅠ	-	-	-	-	-	-	-	0.9	0.9	0.9	0.9	0.9	0.9	0.9	0.8	0.8	0.8	0.8	0.8	0.8	0.8
BSA	-	-	-	-	-	-	-	1	1	1	1	1	1	1	1	1	1	1	1	1	1
dH2O	-	-	-	-	-	-	-	5	5	5	5	5	5	5	5	5	5	5	5	5	5
TOTAL	3	3	3	3	3	3	1	10	10	10	10	10	10	10	10	10	10	10	10	10	10

--->Gel Check

Sample No	1~6	7
Sample DNA	3	1
Loading Dye	1	1
dH2O	2	4
TOTAL	6	6

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]

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	1	2	3
Sample DNA	3	3	3
EcoRⅠ	-	0.1	0.1
SpeⅠ	-	-	0.1
EcoRⅠ Buffer	-	0.9	0.8
BSA	-	1	1
dH2O	-	5	5
TOTAL	3	10	10

--->Gel Check

Sample No	1	2	3
Sample DNA	3	10	10
Loading Dye	1	2	2
dH2O	2	-	-
TOTAL	6	12	12

From left;

BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK
BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Single Digestion -> OK
BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Double Digestion -> Bad

Week 2

24 August, 2008

(22/8)--->Mini prep

BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)

--->Digestion test

BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)

Sample No	5	6	7	8
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	0.1	0.1	0.1	0.1
Buffer 4	0.9	0.9	0.9	0.9
dH2O	8	8	8	8
TOTAL	10	10	10	10

--->Gel Check

Sample No	1~8
Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> ?
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> None?

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

Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)

Sample No	1	2	3	4
Sample DNA	5	5	1	1
EcoRⅠ	0.5	o.5	-	-
SpeⅠ	0.5	0.5	-	-
Buffer 1	1	1	-	-
Buffer 3	-	-	1	-
BSA	1	1	-	-
dH2O	2	2	7.5	-
TOTAL	10	10	10	1

--->Gel Check

Sample No	1~3	4
Sample DNA	10	1
Loading Dye	2	1
dH2O	-	4
TOTAL	12	6

From right;

Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)

--->Digestion (Double Digestion)

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

]

</tr>

Sample No	1	2	3
Sample DNA	33ng/μl×60μl	33ng/μl×60μl	100ng/μl×20μl
PstⅠ	2	2	2
XbaⅠ	2	2	-
SpeⅠ	-	-	2
Buffer 2	-	-	5
Buffer 3	10	10	-
BSA	10	10	5
dH2O	16	16	16
TOTAL	100	100	52

26 August, 2008

(8/25)--->Gel Check

Sample No	1~3
Sample DNA	30
Loading Dye	6
TOTAL	36

1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]

Insert:609bp -> OK

2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]

Insert:609bp -> OK

3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)

-> OK

--->Gel Extract

--->Zymo Clean

5μl
5μl
27μl

--->Gel Check

Sample No	1~3
Sample DNA	2
Loading Dye	2
H2O	8
TOTAL	12

From left;

insert:C0178 -> OK
insert:C0170 -> OK?
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

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	-	-	2	2	2
ligase	1	1	1	1	1
Buffer	2	2	2	2	2
dH2O	6	6	5	4	4
TOTAL	10	10	10	10	10

--->Transformation

insert①C0178
insert②C0170
vector③J04500
insert①C0178+vector③J04500
insert②C0170+vector③J04500

27 August, 2008

Digestion Test

Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]

Sample No	1	2	3
Sample DNA	1	1	1
PstⅠ	-	-	0.5
EcoRⅠ	-	0.5	0.5
Buffer 3	-	1	1
BSA	-	-	1
dH2O	-	7.5	2
TOTAL	1	10	10

--->Gel Check

Sample No	1	2	3
Sample DNA	1	5	5
Loading Dye	1	1	1
H2O	4	-	-
TOTAL	6	6	6

From left;

BBa_R0010
Single Digestion
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

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

insert:C0170 + vector:J04500
insert:C0178 + vector:J04500

--->Digestion Test

insert:C0170 + vector:J04500
insert:C0178 + vector:J04500

Sample No	3	4	5	6
insert:C0170 + vector:J04500	1	-	1	-
insert:C0178 + vector:J04500	-	1	-	1
EcoRⅠ	0.1	0.1	0.1	0.1
SpeⅠ	-	-	0.1	0.1
Buffer 3	1	1	1	1
BSA	-	-	1	1
dH2O	7.8	7.8	6.8	6.8
TOTAL	10	10	10	10

--->(29/8)Gel Check

Sample No	1,2	3~6
Sample DNA	1	10
Loading Dye	1	2
H2O	4	-
TOTAL	6	12

From left;

　-> OK
　-> OK
　-> OK
　-> OK
　-> OK
　-> OK

29 August, 2008

--->Digestion Test

BBa_R0010(2007) [http://partsregistry.org/Part:BBa_R0010]
BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
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_S03160(2006) [http://partsregistry.org/Part:BBa_S03160]

Sample No	S1~10	W1	W2~10
Sample DNA	1	4	2
PstⅠ	-	0.075	0.1
EcoRⅠ	0.1	0.075	0.1
Buffer 3	1	0.75	1
BSA	-	0.75	1
dH2O	7.9	4.35	5.8
TOTAL	10	10	10

--->Gel Check

Sample No.	1~10	S1~S10,W1~W10
Sample DNA	1	10
Loading Dye	1	2
TOTAL	6	12

From left;

1~5,S1,S5,W1~W5

From left;

6~10,S6~S10,W6~W10

(27/8)--->Gel Check

Sample DNA	0.5
Loading Dye	1
dH2O	4.5
TOTAL	6μL

R0010 -> OK

Digestion

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

Sample No	1~3	4
Sample DNA	100ng/μL×20	200ng/μL×30
PstⅠ	2	2
XbaⅠ	2	-
SpeⅠ	-	2
Buffer 2	-	5
Buffer 3	5	-
BSA	5	5
dH2O	16	6
TOTAL	50	50

--->Gel Check

Sample No.	1~3	4
Sample DNA	10	3
Loading Dye	2	2
dH2O	-	7
TOTAL	12	12

insert-I9026 : 685bp -> None

insert-I9030 : 745bp -> None

insert-S03154 : 685bp -> None

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

insert:C0170 + vector:J04500
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 : 10~18	Double : 19~27
Sample DNA	1	5
XbaⅠ	0.1	0.1
SpeⅠ	-	0.1
Buffer 2	1	1
BSA	1	1
dH2O	6.9	2.8
TOTAL	10	10

--->Gel Check

Sample No	1~9	10~18	19~27
Sample DNA	1	10	10
Loading Dye	1	2	2
dH2O	4	-	-
TOTAL	6	12	12

From left;

Sanple No.1~13

-> OK

From left;

Sample No.14~17,24~16

14 -> OK

15,16 -> Bad

17 -> None

24~16 -> Bad

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

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

Sample No	1~3	4
Sample DNA	12	10
PstⅠ	0.2	0.2
XbaⅠ	0.2	-
SpeⅠ	-	0.4
Buffer 2	-	1.5
Buffer 3	2	-
BSA	2	1.5
dH2O	3.6	1.4
TOTAL	20	15

--->(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 2~5	Double Digestion 2~5	Single Digestion T9002①	Single Digesiton T9002②	<Double Digestion T9002①②
Sample DNA	1	3	3	1	3
XbaⅠ	0.1	0.1	0.1	0.1	0.1
SpeⅠ	-	0.1	-	-	0.1
Buffer 2	0.9	0.8	0.9	0.9	0.8
BSA	1	1	1	1	1
dH2O	7	5	5	7	5
TOTAL	10	10	10	10	10

--->Gel Check

Sample No	Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①②	Single & Double Digestion
Sample DNA	1	10
Loading Dye	1	2
dH2O	4	-
TOTAL	6	12

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

Sample No	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24	18

From left;

insert-1(I9026)

insert-2(I9030)

insert-3(S03154)

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

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

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

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

From left;

Plac+RBS+CinI+LVA

C1,C2 -> OK

C3 -> Bad

C4~C6 -> OK

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

Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

S03154 -> OK
R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK

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	Single	Double
Sample DNA	1	3
XbaⅠ	0.1	0.1
PstⅠ	0.1	0.1
Buffer 2	0.9	-
Buffer 3	-	-0.8
BSA	1	1
dH2O	7	5
TOTAL	10	10

--->Gel Check

Sample DNA	10
Loading Dye	2
TOTAL	12

From left;

Single Digestion : R1,R3~R7 -> OK
Single Digestion : C1,C2,C4~C8 -> OK

From left;

Double Digestion : R1,R3~R7 -> OK
Double Digestion : C1,C2,C4~C8 -> OK

(2/9)--->Phenotype-test : mix

Sample No.	1	2	3	4	5	6	7	8	9	10	11
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]	2	2	2	2	1	1	1	1	2	1	1
AHL	1	1	1	1	1	1	1	1	1	1	1

|

| 8h

V

Spindown

|

V

the intensity GFP

4 September, 2008

--->Gel Check

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

From left;

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

From left;

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

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

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

Sample No	1~3
Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

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

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

Sample No	①	②	③	④
Sample DNA	5	5	1	1
EcoRⅠ	0.5	o.5	-	-
SpeⅠ	0.5	0.5	-	-
Buffer 1	1	1	-	-
Buffer 3	-	-	1	-
BSA	1	1	-	-
dH2O	2	2	7.5	-
TOTAL	10	10	10	1

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]

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

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

Cultured transformants(LB,37℃,14h)

BBa_K084007~BBa_K084009
BBa_T9002
BBa_S03623

cultured above in new LB medium.
-->mix(16/9)

16 September, 2008

(15/9)->mix cultures in the way prescribed in the Table below.

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

ホーム	メンバー紹介	プロジェクト紹介	Parts Submitted to the Registry	モデリング	ノート

Team:Chiba/jk/β

From 2008.igem.org

Contents

Week 1

17 August, 2008

18 August, 2008

19 August, 2008

20 August, 2008

21 August, 2008

22 August, 2008

23 August, 2008

Week 2

24 August, 2008

25 August, 2008

26 August, 2008

27 August, 2008

28 August, 2008

29 August, 2008

30 August, 2008

Week 3

31 August, 2008

1 September, 2008

2 September, 2008

3 September, 2008

4 September, 2008

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

Week 5

14 September, 2008

15 September, 2008

16 September, 2008