"

 

From 2008.igem.org

β班の実験ノート

Contents 1 Week 1 1.1 17 August, 2008 1.2 18 August, 2008 1.3 19 August, 2008 1.4 20 August, 2008 1.5 21 August, 2008 1.6 22 August, 2008 1.7 23 August, 2008 2 Week 2 2.1 24 August, 2008 2.2 25 August, 2008 2.3 26 August, 2008 2.4 27 August, 2008 2.5 28 August, 2008 2.6 29 August, 2008 2.7 30 August, 2008 3 Week 3 3.1 31 August, 2008 3.2 1 September, 2008 3.3 2 September, 2008 3.4 3 September, 2008 3.5 4 September, 2008 3.6 5 September, 2008 3.7 6 September, 2008 4 Week 4 4.1 7 September, 2008 4.2 8 September, 2008 4.3 9 September, 2008 4.4 10 September, 2008 4.5 11 September, 2008 4.6 12 September, 2008 4.7 13 September, 2008 5 Week 5 5.1 14 September, 2008 5.2 15 September, 2008 5.3 16 September, 2008

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

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	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

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	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)

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	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

1. 5μl
2. 5μl
3. 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

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	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

1. insert:C0170 + vector:J04500
2. insert:C0178 + vector:J04500

--->Digestion Test

1. insert:C0170 + vector:J04500
2. 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

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	S1~10	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

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	1~3	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

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 : 10~18	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

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	1~3	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

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 September, 2008

(2/9)--->Mini prep

--->Gel Check

Sample DNA 1 Loading Dye 1 dH2O 4 TOTAL 6 From left; S03154 R1,R3~R7

From left; C1,C2,C4~C8

--->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

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

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

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 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	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

16 September, 2008

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