Team:Chiba/jk/β

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β班の実験ノート

Week 1

17 August, 2008

Transformation

BBa_R0040(2008) [1] -> No colonies on plates
BBa_C0070(2008) [2] -> No colonies on plates
BBa_C0076(2008) [3] -> No colonies on plates
BBa_C0077(2008) [4] -> No colonies on plates
BBa_C0078(2008) [5] -> No colonies on plates
BBa_C0079(2008) [6] -> No colonies on plates

18 August, 2008P

PCR

BBa_C0070(2008) [7]
BBa_C0076(2008) [8]
BBa_C0077(2008) [9]
BBa_C0078(2008) [10]
BBa_C0079(2008) [11]
BBa_F2620(2007) [12]･･･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) [13] ->None
BBa_C0076(2008) [14] ->None
BBa_C0077(2008) [15] ->None
BBa_C0078(2008) [16] ->None
BBa_C0079(2008) [17] ->None
BBa_F2620(2007) [18]･･･control ->OK

Transformation

compitent cell : XL10G

BBa_C0070(2007) [19]
BBa_C0070(2006) [20]
BBa_C0076(2007) [21]
BBa_C0076(2006) [22] -> No colonies on plates(19/8)
BBa_C0078(2007) [23]
BBa_C0078(2006) [24]
BBa_C0170(2007) [25]
BBa_C0170(2006) [26]
BBa_C0178(2007) [27]
BBa_C0178(2006) [28]

--->(20/8)Mini prep

19 August, 2008

PCR

BBa_I9026(2007) [29]
BBa_I9026(2006) [30]
BBa_I9030(2007) [31]
BBa_I9030(2006) [32]
BBa_S3154(2007) [33]
BBa_S3154(2006) [34]
BBa_F2620(2007) [35]･･･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) [36] ->None
BBa_I9026(2006) [37] ->None
BBa_I9030(2007) [38] ->None
BBa_I9030(2006) [39] ->None
BBa_S3154(2007) [40] ->None
BBa_S3154(2006) [41] ->None
BBa_F2620(2007) [42]･･･control ->OK

20 August, 2008

(18/8)--->Mini prep

BBa_C0070(2007) [43]
BBa_C0070(2006) [44]
BBa_C0076(2007) [45]
BBa_C0078(2007) [46]
BBa_C0078(2006) [47]
BBa_C0170(2007) [48]
BBa_C0170(2006) [49]
BBa_C0178(2007) [50]
BBa_C0178(2006) [51]

--->Gel Check

Sample DNA	3
Loading Dye	2
dH2O	7
TOTAL	12μL

From left;

BBa_C0076(2007) [52] -> None
BBa_C0078(2007) [53] -> None
BBa_C0078(2006) [54] -> None

Sample DNA	10
Loading Dye	4
dH2O	10
TOTAL	24μL

From left;

BBa_C0078(2007) [55] -> None
BBa_C0078(2006) [56] -> OK
BBa_C0076(2007) [57] -> None
BBa_C0070(2007) [58] -> OK
BBa_C0070(2006) [59] -> OK

Sample DNA	3
Loading Dye	2
dH2O	7
TOTAL	12μL

From left;

BBa_C0170(2007) [60] -> OK
BBa_C0170(2006) [61] -> OK
BBa_C0178(2007) [62] -> OK
BBa_C0178(2006) [63] -> OK

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

BBa_C0170(2007) [64]
BBa_C0170(2006) [65]
BBa_C0178(2007) [66]
BBa_C0178(2006) [67]

Sample No	1	2	3	4	5	6	7	8
BBa_C0170(2006) [68]	3	-	-	-	3	-	-	-
BBa_C0170(2007) [69]	-	3	-	-	-	3	-	-
BBa_C0178(2006) [70]	-	-	3	-	-	-	3	-
BBa_C0178(2007) [71]	-	-	-	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) [72] -> OK
BBa_C0170(2007) [73] -> OK
BBa_C0178(2006) [74] -> OK
BBa_C0178(2007) [75] -> OK

(Double Digestion 1~4)

BBa_C0170(2006) [76] -> OK
BBa_C0170(2007) [77] -> OK
BBa_C0178(2006) [78] -> OK
BBa_C0178(2007) [79] -> OK

21 August, 2008

(20/8)--->PCR

BBa_C0070(2007) [80]
BBa_C0070(2006) [81]
BBa_C0076(2007) [82]
BBa_C0078(2007) [83]
BBa_C0078(2006) [84]

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) [85] -> None
BBa_C0078(2006) [86] -> None
BBa_C0076(2007) [87] -> None
BBa_C0070(2007) [88] -> OK
BBa_C0070(2006) [89] -> OK

Transformation

BBa_S03154(2007) [90]
BBa_S03154(2006) [91]
BBa_I9026(2007) [92]
BBa_I9026(2006) [93]
BBa_I9030(2007) [94]
BBa_I9030(2006) [95]

Compitentcell : XL10G

--->(23/8)Digestion

22 August, 2008

Transformation

BBa_J13002(2007) [96]
BBa_J13002(2006) [97]
BBa_J04500(2007) [98]
BBa_J04500(2006) [99]

(Locate & 2 Punch ~iyama ver.~)

BBa_C0076(2008) [100] -> No colonies on plates
BBa_C0077(2008) [101] -> No colonies on plates
BBa_C0078(2008) [102] -> No colonies on plates

23 August, 2008

--->Mini prep

BBa_S03154(2007) [103]
BBa_S03154(2006) [104]
BBa_I9026(2007) [105]
BBa_I9026(2006) [106]
BBa_I9030(2007) [107]
BBa_I9030(2006) [108]

(21/8)--->Digestion

BBa_S03154(2007) [109]
BBa_S03154(2006) [110]
BBa_I9026(2007) [111]
BBa_I9026(2006) [112]
BBa_I9030(2007) [113]
BBa_I9030(2006) [114]
BBa_J04500(2007) [115]

--->Gel Check</table>

From the left;
①BBa_C0170(2007) [116] -> OK
②BBa_C0170(2006) [117] -> OK
③BBa_C0178(2007) [118] -> OK
④BBa_C0178(2006) [119] -> OK

BBa_S03154(2007) [120]
BBa_S03154(2006) [121]
BBa_I9026(2007) [122]
BBa_I9026(2006) [123]
BBa_I9030(2007) [124]
BBa_I9030(2006) [125]
BBa_J04500(2007) [126]

BBa_S03154(2007) [127]
BBa_S03154(2006) [128]
BBa_I9026(2007) [129]
BBa_I9026(2006) [130]
BBa_I9030(2007) [131]
BBa_I9030(2006) [132]
BBa_J04500(2007) [133]
M
BBa_S03154(2007) [134]
BBa_S03154(2006) [135]
BBa_I9026(2007) [136]
BBa_I9026(2006) [137]
BBa_I9030(2007) [138]
BBa_I9030(2006) [139]
BBa_J04500(2007) [140]

BBa_J04500(2007) [141]
BBa_J04500(2007) [142]Single Digestion
BBa_J04500(2007) [143]Double Digestion

Week 2

24 August, 2008

(22/8)--->Mini prep

BBa_J13002[144](2007)
BBa_J13002[145](2006)
BBa_J04500[146](2007)
BBa_J04500[147](2006)

--->Digestion

BBa_J13002[148](2007)
BBa_J13002[149](2006)
BBa_J04500[150](2007)
BBa_J04500[151](2006)

--->Gel Check

Sample No	①	②	③	④	⑤	⑥	⑦	⑧
Sample DNA	1	1	1	1	1	1	1	1
Loading Dye	1	1	1	1	1	1	1	1
dH2O	4	4	4	4	4	4	4	4
TOTAL	6	6	6	6	6	6	6	6

From the left;

⑤BBa_J04500[152](2007)

⑥BBa_J04500[153](2006)

⑦BBa_J13002[154](2007)

⑧BBa_J13002[155](2006)

（↓single digestuion）

⑤BBa_J04500[156](2007)

⑥BBa_J04500[157](2006)

⑦BBa_J13002[158](2007)

⑧BBa_J13002[159](2006)

Sample No	⑤	⑥	⑦	⑧
Sample DNA	9	9	9	9
Loading Dye	2	2	2	2
dH2O	1	1	1	1
TOTAL	12	12	12	12

From the left; （single digestuion）

⑤BBa_J04500[160](2007)

⑥BBa_J04500[161](2006)

⑦BBa_J13002[162](2007)

⑧BBa_J13002[163](2006)

25 August, 2008

(24/8)--->Digestion

①Double Digestion:BBa_J04500[164](2007)

②Double Digesiton:BBa_R0010[165](2007)

③Single Digestion:BBa_R0010[166](2007)

④BBa_R0010[167](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	②	③	④
Sample DNA	10	10	10	10
Loading Dye	2	2	2	1
dH2O	-	-	-	4
TOTAL	12	12	12	6

From right;

①Double Digestion:BBa_J04500[168](2007) -> OK

②Double Digesiton:BBa_R0010[169](2007) -> ?(low)

③Single Digestion:BBa_R0010[170](2007) -> ?(low)

④BBa_R0010[171](2007) -> OK(low)

--->Digestion

①BBa_C0178[172]

②BBa_C0170[173]

③BBa_J04500[174](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

--->Gel Check

Sample No	①	②	③
Sample DNA	30	30	30
Loading Dye	6	6	6
TOTAL	36	36	36

①BBa_C0178[175]

Insert:609bp -> OK

②BBa_C0170[176]

Insert:609bp -> OK

③BBa_J04500[177](2007)

-> OK

26 August, 2008

(8/25)--->Gel Extract

--->Zymo Clean

①5μl

②5μl

③27μl

--->Gel Check

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

From left;

①insert:C0178 -> OK

②insert:C0170 -> OK?

③Vector:J04500 -> OK

--->Dephosphorylation

③

--->Zymo Clean

③->9μl

--->Gel Check

Sample DNA ③	1
Loading Dye	1
dH2O	4
TOTAL	6

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[178]

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;

①BBa_R0010

②Single Digestion

③Double Digestion

(26/8)--->PCR

Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[179](2007)).

]

</tr>

Sample No	(4)-1~5	(5)-1~5	(F2620)
insert①C0178	1	-	-
insert②C0170	-	1	-
vector③J04500	-	-	2
ligase	1	1	1
Buffer	2	2	2
dH2O	6	6	5
TOTAL	10	10	10

--->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)Mini prepDigestion Test

Transformation comptent cell : XL10G

BBa_C0062(2007) [180]
BBa_C0062(2006) [181] -> no colony on plates
BBa_C0179(2007) [182]
BBa_C0179(2006) [183]
BBa_S03156(2007) [184]
BBa_S03156(2006) [185]
BBa_S03158(2007) [186]
BBa_S03158(2006) [187]
BBa_S03160(2007) [188]
BBa_S0316(2006) [189]

--->Mini prep

--->(29/8)Gel Check

28 August, 2008]

(27/8)--->Mini prep

①insert:C0170 + vector:J04500

②insert:C0178 + vector:J04500

--->Digestion Test

--->(29/8)Gel Check

Sample No	①②	Single Digesiton:①②	Double Digesiton:①②
Sample DNA	1	10	10
Loading Dye	1	2	2
H2O	4	-	-
TOTAL	6	12	12

From left;

①　-> OK

②　-> OK

Single Digestion:①　-> OK

Single Digestion:②　-> OK

Double Digestion:①　-> OK

Double Digestion:②　-> OK

29 August, 2008

--->Digestion Test

①BBa_R0010(2007) [190]

②BBa_C0062(2007) [191]

③BBa_C0179(2007) [192]

④BBa_C0179(2006) [193]

⑤BBa_S03156(2007) [194]

⑥BBa_S03156(2006) [195]

⑦BBa_S03158(2007) [196]

⑧BBa_S03158(2006) [197]

⑨BBa_S03160(2007) [198]

⑩BBa_S03160(2006) [199]

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 DNA	10
Loading Dye	2
TOTAL	12

From left;
①~⑤,S①⑤,W①~⑤

From left;
⑥~⑩,S⑥~⑩,W⑥~⑩

(27/8)--->

R0010

Digestion

①BBa_I9026(2007) [200]

②BBa_I9030(2007) [201]

③BBa_S03154(2007) [202]

④BBa_R0010(2006) [203]

Sample No	S①~③	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 DNA	10	3
Loading Dye	2	2
dH2O	-	7
TOTAL	12

From left;

From left;

From left;

From left;

)--->Gel Extract

--->Zymo Clean

=>1.5μL/2colum

--->SAP

④

--->Zymo Clean

---> miss!

30 August, 2008

Transformation comptent cell : XL10G

BBa_I9026(2007) [204]
BBa_I9030(2006) [205]
BBa_S03154(2007) [206]
BBa_S03156(2007) [207]
BBa_S03158(2007) [208]
BBa_S03160(2007) [209]
BBa_C0062(2007) [210]
BBa_C0179(2007) [211]

--->(31/8)Mini prep

(29/8)--->Mini prep

①insert:C0170 + vector:J04500

②insert:C0178 + vector:J04500

--->Digestion Test

--->Gel Check

From left;

From left;

From left;

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

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, 2008P

19 August, 2008

20 August, 2008

21 August, 2008

22 August, 2008

23 August, 2008

Week 2

Week 3

Week 4