Team:Chiba/jk/β
From 2008.igem.org
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
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
|
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
|
|
|
--->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
|
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
|
- 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]
- From left; Sanple No. 1~7
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 CheckSample 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
- From left;
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?
- From left;
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)
- From right;
--->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)
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 </tr>
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
- 1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
- 2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
- Insert:609bp -> OK
- 2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
- 3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
- -> OK
- 3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
--->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
- From left;
- --->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
Sample No (1) (2) (3) (4) (5) insert①C0178 1 - - 1 - insert②C0170 - 1 - - 1 vector③J04500 - - 2 2 2 ligase 1 1 1 1 1 </tr>
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
- 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
- From left;
- (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
- From left;
- --->(28/8)Miniprep
- 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
- From left;
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;
- 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
- 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;
- 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
- 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 CheckSample 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
- From Left
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)
- From left;
- insert-3(S03154)
- vector-4(R0010)
- --->zymo
- vector-4(R0010)
- -> 15μL
- --->SAP
- vectr-4(R0010)
- --->Zymo
- 1 -> 7μL
- 2 -> 7
- 3 -> 7
- 4 -> 20
- 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
- From left;
- (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 CheckSample 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;
- 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
- 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;
- 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;
- 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
- 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;
- 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
16 September, 2008
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- --->Zymo Clean