Team:Chiba/jk/β

From 2008.igem.org

(Difference between revisions)

Revision as of 13:45, 11 September 2008

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, 2008

Transformation

BBa_C0070(2007) [7]
BBa_C0070(2006) [8]
BBa_C0076(2007) [9]
BBa_C0076(2006) [10] -> No colonies on plates
BBa_C0078(2007) [11]
BBa_C0078(2006) [12]
BBa_C0170(2007) [13]
BBa_C0170(2006) [14]
BBa_C0178(2007) [15]
BBa_C0178(2006) [16]

--->(20/8)Mini prep

19 August, 2008

PCR

BBa_I9026(2007) [17]
BBa_I9026(2006) [18]
BBa_I9030(2007) [19]
BBa_I9030(2006) [20]
BBa_S3154(2007) [21]
BBa_S3154(2006) [22]
BBa_F2620(2007) [23]･･･control

DNA Template	1
dNTP mix	10
Foward Primer	5
Reverse Primer	5
DNA polymerase	1
Thermopol Buffer	10
dH2O	68
TOTAL	100

--->Gel Check

Sample DNA	3
Loading Dye	2
dH2O	7
TOTAL	12

BBa_I9026(2007) [24] ->None
BBa_I9026(2006) [25] ->None
BBa_I9030(2007) [26] ->None
BBa_I9030(2006) [27] ->None
BBa_S3154(2007) [28] ->None
BBa_S3154(2006) [29] ->None
BBa_F2620(2007) [30]･･･control ->OK

20 August, 2008

(18/8)--->Mini prep

BBa_C0070(2007) [31]
BBa_C0070(2006) [32]
BBa_C0076(2007) [33]
BBa_C0078(2007) [34]
BBa_C0078(2006) [35]
BBa_C0170(2007) [36]
BBa_C0170(2006) [37]
BBa_C0178(2007) [38]
BBa_C0178(2006) [39]

--->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 the left;
①BBa_C0170(2007) [40] -> OK
②BBa_C0170(2006) [41] -> OK
③BBa_C0178(2007) [42] -> OK
④BBa_C0178(2006) [43] -> OK

BBa_C0170(2007) [44] -> OK
BBa_C0170(2006) [45] -> OK
BBa_C0178(2007) [46] -> OK
BBa_C0178(2006) [47] -> OK

BBa_C0076(2007) [48] -> None
BBa_C0078(2007) [49] -> None
BBa_C0078(2006) [50] -> None

BBa_C0078(2007) [51] -> None
BBa_C0078(2006) [52] -> OK
BBa_C0076(2007) [53] -> None
BBa_C0070(2007) [54] -> OK
BBa_C0070(2006) [55] -> OK

--->Digestion

BBa_C0070(2007) [56]
BBa_C0070(2006) [57]
BBa_C0078(2007) [58]
BBa_C0078(2006) [59]

21 August, 2008

(20/8)--->PCR

BBa_C0070(2007) [60]
BBa_C0070(2006) [61]
BBa_C0076(2007) [62]
BBa_C0078(2007) [63]
BBa_C0078(2006) [64]

--->Gel Check

From the left;

BBa_C0078(2007) [65] -> None
BBa_C0078(2006) [66] -> None
BBa_C0076(2007) [67] -> None
BBa_C0070(2007) [68] -> OK
BBa_C0070(2006) [69] -> OK

Transformation

BBa_S03154(2007) [70]
BBa_S03154(2006) [71]
BBa_I9026(2007) [72]
BBa_I9026(2006) [73]
BBa_I9030(2007) [74]
BBa_I9030(2006) [75]

22 August, 2008

Transformation

BBa_J13002(2007) [76]
BBa_J13002(2006) [77]
BBa_J04500(2007) [78]
BBa_J04500(2006) [79]

(Locate & 2 Punch ~iyama ver.~)

BBa_C0076(2008) [80] -> No colonies on plates
BBa_C0077(2008) [81] -> No colonies on plates
BBa_C0078(2008) [82] -> No colonies on plates

23 August, 2008

--->Mini prep

BBa_S03154(2007) [83]
BBa_S03154(2006) [84]
BBa_I9026(2007) [85]
BBa_I9026(2006) [86]
BBa_I9030(2007) [87]
BBa_I9030(2006) [88]

--->Digestion

BBa_S03154(2007) [89]
BBa_S03154(2006) [90]
BBa_I9026(2007) [91]
BBa_I9026(2006) [92]
BBa_I9030(2007) [93]
BBa_I9030(2006) [94]
BBa_J04500(2007) [95]

--->Gel Check</table>

From the left;
①BBa_C0170(2007) [96] -> OK
②BBa_C0170(2006) [97] -> OK
③BBa_C0178(2007) [98] -> OK
④BBa_C0178(2006) [99] -> OK

BBa_S03154(2007) [100]
BBa_S03154(2006) [101]
BBa_I9026(2007) [102]
BBa_I9026(2006) [103]
BBa_I9030(2007) [104]
BBa_I9030(2006) [105]
BBa_J04500(2007) [106]

BBa_S03154(2007) [107]
BBa_S03154(2006) [108]
BBa_I9026(2007) [109]
BBa_I9026(2006) [110]
BBa_I9030(2007) [111]
BBa_I9030(2006) [112]
BBa_J04500(2007) [113]
M
BBa_S03154(2007) [114]
BBa_S03154(2006) [115]
BBa_I9026(2007) [116]
BBa_I9026(2006) [117]
BBa_I9030(2007) [118]
BBa_I9030(2006) [119]
BBa_J04500(2007) [120]

BBa_J04500(2007) [121]
BBa_J04500(2007) [122]Single Digestion
BBa_J04500(2007) [123]Double Digestion

Week 2

24 August, 2008

(22/8)--->Mini prep

BBa_J13002[124](2007)
BBa_J13002[125](2006)
BBa_J04500[126](2007)
BBa_J04500[127](2006)

--->Digestion

BBa_J13002[128](2007)
BBa_J13002[129](2006)
BBa_J04500[130](2007)
BBa_J04500[131](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[132](2007)

⑥BBa_J04500[133](2006)

⑦BBa_J13002[134](2007)

⑧BBa_J13002[135](2006)

（↓single digestuion）

⑤BBa_J04500[136](2007)

⑥BBa_J04500[137](2006)

⑦BBa_J13002[138](2007)

⑧BBa_J13002[139](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[140](2007)

⑥BBa_J04500[141](2006)

⑦BBa_J13002[142](2007)

⑧BBa_J13002[143](2006)

25 August, 2008

(24/8)--->Digestion

①Double Digestion:BBa_J04500[144](2007)

②Double Digesiton:BBa_R0010[145](2007)

③Single Digestion:BBa_R0010[146](2007)

④BBa_R0010[147](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[148](2007) -> OK

②Double Digesiton:BBa_R0010[149](2007) -> ?(low)

③Single Digestion:BBa_R0010[150](2007) -> ?(low)

④BBa_R0010[151](2007) -> OK(low)

--->Digestion

①BBa_C0178[152]

②BBa_C0170[153]

③BBa_J04500[154](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[155]

Insert:609bp -> OK

②BBa_C0170[156]

Insert:609bp -> OK

③BBa_J04500[157](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[158]

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[159](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) [160]
BBa_C0062(2006) [161] -> no colony on plates
BBa_C0179(2007) [162]
BBa_C0179(2006) [163]
BBa_S03156(2007) [164]
BBa_S03156(2006) [165]
BBa_S03158(2007) [166]
BBa_S03158(2006) [167]
BBa_S03160(2007) [168]
BBa_S0316(2006) [169]

--->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) [170]

②BBa_C0062(2007) [171]

③BBa_C0179(2007) [172]

④BBa_C0179(2006) [173]

⑤BBa_S03156(2007) [174]

⑥BBa_S03156(2006) [175]

⑦BBa_S03158(2007) [176]

⑧BBa_S03158(2006) [177]

⑨BBa_S03160(2007) [178]

⑩BBa_S03160(2006) [179]

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) [180]

②BBa_I9030(2007) [181]

③BBa_S03154(2007) [182]

④BBa_R0010(2006) [183]

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) [184]
BBa_I9030(2006) [185]
BBa_S03154(2007) [186]
BBa_S03156(2007) [187]
BBa_S03158(2007) [188]
BBa_S03160(2007) [189]
BBa_C0062(2007) [190]
BBa_C0179(2007) [191]

--->(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

@@ Line 40: / Line 40: @@
 ::*BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154]
 ::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">DNA Template</td>
+<td>1</td>
+</tr>
+<tr>
+<td>dNTP mix</td>
+<td>10</td>
+</tr>
+<tr>
+<td>Foward Primer</td>
+<td>5</td>
+</tr>
+<tr>
+<td>Reverse Primer</td>
+<td>5</td>
+</tr>
+<tr>
+<td>DNA polymerase</td>
+<td>1</td>
+</tr>
+<tr>
+<td>Thermopol Buffer</td>
+<td>10</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>68</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>100</td>
+</tr>
+</table>
 :--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample DNA</td>
+<td>3</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>7</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td>
+</tr>
+</table>
 {|align="justify"
 |[[Image:Chiba-0818.JPG]]

Team:Chiba/jk/β

From 2008.igem.org

Revision as of 13:45, 11 September 2008

Contents

Week 1

Week 2

Week 3

Week 4