Team:Chiba/jk/β/weaks 1-2

From 2008.igem.org

>送受信班

Week 1

17 August, 2008

Transformation

competent cells : XL10G

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, 2008

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

competent cells : 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

--->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

competent cells : XL10G

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

--->(23/8)Digestion

22 August, 2008

Transformation

competent cells : XL10G

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
BBa_J04500(2008) [103] -> No colonies on plates

--->(24/8)Mini prep

23 August, 2008

(21/8)--->Mini prep

BBa_S03154(2007) [104]
BBa_S03154(2006) [105]
BBa_I9026(2007) [106]
BBa_I9026(2006) [107]
BBa_I9030(2007) [108]
BBa_I9030(2006) [109]

--->Digestion test

BBa_S03154(2007) [110]
BBa_S03154(2006) [111]
BBa_I9026(2007) [112]
BBa_I9026(2006) [113]
BBa_I9030(2007) [114]
BBa_I9030(2006) [115]
BBa_J04500(2007) [116]

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) [117]	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-
BBa_I9030(2006) [118]	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-
BBa_S03154(2007) [119]	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-
BBa_S03154(2006) [120]	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-
BBa_I9026(2007) [121]	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-
BBa_I9026(2007) [122]	-	-	-	-	-	3	-	-	-	-	-	-	3	-	-	-	-	-	-	3	-
BBa_J04500(2007) [123]	-	-	-	-	-	-	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) [124]
BBa_S03154(2006) [125]
BBa_I9026(2007) [126]
BBa_I9026(2006) [127]
BBa_I9030(2007) [128]
BBa_I9030(2006) [129]
BBa_J04500(2007) [130]

Sample No	8~21
Sample DNA	10
Loading Dye	2
TOTAL	12

From left;

(Single Digestion) Sanple No. 8~14

BBa_S03154(2007) [131] -> OK
BBa_S03154(2006) [132] -> OK
BBa_I9026(2007) [133] -> OK
BBa_I9026(2006) [134] -> OK
BBa_I9030(2007) [135] -> OK
BBa_I9030(2006) [136] -> OK
BBa_J04500(2007) [137] -> OK

(Double Digestion) Sample No. 15~21

BBa_S03154(2007) [138] -> OK
BBa_S03154(2006) [139] -> OK
BBa_I9026(2007) [140] -> OK
BBa_I9026(2006) [141] -> OK
BBa_I9030(2007) [142] -> OK
BBa_I9030(2006) [143] -> OK
BBa_J04500(2007) [144] -> OK?

Digestion test

BBa_J04500(2007:old-sample)[145]

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) [146] -> OK
BBa_J04500(2007) [147]Single Digestion -> OK
BBa_J04500(2007) [148]Double Digestion -> Bad

Week 2

24 August, 2008

(22/8)--->Mini prep

BBa_J04500[149](2007)
BBa_J04500[150](2006)
BBa_J13002[151](2007)
BBa_J13002[152](2006)

--->Digestion test

BBa_J04500[153](2007)
BBa_J04500[154](2006)
BBa_J13002[155](2007)
BBa_J13002[156](2006)

Sample No	5	6	7	8
BBa_J04500[157](2007)	1	-	-	-
BBa_J04500[158](2006)	-	1	-	-
BBa_J13002[159](2007)	-	-	1	-
BBa_J13002[160](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[161](2007) -> ?
BBa_J04500[162](2006) -> OK
BBa_J13002[163](2007) -> OK
BBa_J13002[164](2006) -> OK
BBa_J04500[165](2007) -> OK
BBa_J04500[166](2006) -> OK
BBa_J13002[167](2007) -> OK
BBa_J13002[168](2006) -> None?

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

From the left; （single digestuion）

5, BBa_J04500[169](2007) -> OK

6, BBa_J04500[170](2006) -> OK

7, BBa_J13002[171](2007) -> 3400bp:Bad, 2200bp:OK

8, BBa_J13002[172](2006) -> OK

25 August, 2008

(24/8)--->Digestion

Double Digestion:BBa_J04500[173](2007)
Double Digesiton:BBa_R0010[174](2007)
Single Digestion:BBa_R0010[175](2007)
BBa_R0010[176](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[177](2007) -> OK
Double Digesiton:BBa_R0010[178](2007) -> ?(low)
Single Digestion:BBa_R0010[179](2007) -> ?(low)
BBa_R0010[180](2007) -> OK(low)

--->Digestion (Double Digestion)

BBa_C0178[181]
BBa_C0170[182]
BBa_J04500[183](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[184]

Insert:609bp -> OK

2, BBa_C0170[185]

Insert:609bp -> OK

3, BBa_J04500[186](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[187]

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[188](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) [189]
BBa_C0062(2006) [190] -> no colony on plates
BBa_C0179(2007) [191]
BBa_C0179(2006) [192]
BBa_S03156(2007) [193]
BBa_S03156(2006) [194]
BBa_S03158(2007) [195]
BBa_S03158(2006) [196]
BBa_S03160(2007) [197]
BBa_S0316(2006) [198]

--->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) [199]
BBa_C0062(2007) [200]
BBa_C0179(2007) [201]
BBa_C0179(2006) [202]
BBa_S03156(2007) [203]
BBa_S03156(2006) [204]
BBa_S03158(2007) [205]
BBa_S03158(2006) [206]
BBa_S03160(2007) [207]
BBa_S03160(2006) [208]

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) [209]
BBa_I9030(2007) [210]
BBa_S03154(2007) [211]
BBa_R0010(2006) [212]

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) [213]
BBa_I9030(2006) [214]
BBa_S03154(2007) [215]
BBa_S03156(2007) [216]
BBa_S03158(2007) [217]
BBa_S03160(2007) [218]
BBa_C0062(2007) [219]
BBa_C0179(2007) [220]

--->(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)[221] -> 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??

Team:Chiba/jk/β/weaks 1-2

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