Team:Chiba/jk/β/week 2

From 2008.igem.org

(Difference between revisions)

Revision as of 11:52, 23 October 2008

>送受信班

>Protocols

Week 2

>last week

24 August, 2008

(22/8)--->Mini prep

BBa_J04500(2007)
BBa_J04500(2006)
BBa_J13002(2007)
BBa_J13002(2006)

--->Digestion test

BBa_J04500(2007)
BBa_J04500(2006)
BBa_J13002(2007)
BBa_J13002(2006)

Sample No.	5	6	7	8
BBa_J04500(2007)	1	-	-	-
BBa_J04500(2006)	-	1	-	-
BBa_J13002(2007)	-	-	1	-
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(2007) -> ?
BBa_J04500(2006) -> OK
BBa_J13002(2007) -> OK
BBa_J13002(2006) -> OK
BBa_J04500(2007) -> OK
BBa_J04500(2006) -> OK
BBa_J13002(2007) -> OK
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(2007) -> OK

6, BBa_J04500(2006) -> OK

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

8, BBa_J13002(2006) -> OK

25 August, 2008

(24/8)--->Digestion

Double Digestion:BBa_J04500[1](2007)
Double Digesiton:BBa_R0010[2](2007)
Single Digestion:BBa_R0010[3](2007)
BBa_R0010[4](2007)

Sample No.	1	2	3	4
Sample DNA	5	5	1	1
EcoRⅠ	0.5	0.5	-	-
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[5](2007) -> OK
Double Digesiton:BBa_R0010[6](2007) -> ?(low)
Single Digestion:BBa_R0010[7](2007) -> ?(low)
BBa_R0010[8](2007) -> OK(low)

--->Digestion (Double Digestion)

BBa_C0178[9]
BBa_C0170[10]
BBa_J04500[11](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
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[12]

Insert:609bp -> OK

2, BBa_C0170[13]

Insert:609bp -> OK

3, BBa_J04500[14](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[15]

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[16](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) [17]
BBa_C0062(2006) [18] -> no colony on plates
BBa_C0179(2007) [19]
BBa_C0179(2006) [20]
BBa_S03156(2007) [21]
BBa_S03156(2006) [22]
BBa_S03158(2007) [23]
BBa_S03158(2006) [24]
BBa_S03160(2007) [25]
BBa_S0316(2006) [26]

--->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) [27]
BBa_C0062(2007) [28]
BBa_C0179(2007) [29]
BBa_C0179(2006) [30]
BBa_S03156(2007) [31]
BBa_S03156(2006) [32]
BBa_S03158(2007) [33]
BBa_S03158(2006) [34]
BBa_S03160(2007) [35]
BBa_S03160(2006) [36]

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) [37]
BBa_I9030(2007) [38]
BBa_S03154(2007) [39]
BBa_R0010(2006) [40]

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) [41]
BBa_I9030(2006) [42]
BBa_S03154(2007) [43]
BBa_S03156(2007) [44]
BBa_S03158(2007) [45]
BBa_S03160(2007) [46]
BBa_C0062(2007) [47]
BBa_C0179(2007) [48]

--->(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)[49] -> 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??

>next week

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 15: / Line 15: @@
 :--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006)
@@ Line 27: / Line 27: @@
 </tr>
 <tr>
-<td>BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)</td>
+<td>[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)</td>
 <td>1</td><td>-</td><td>-</td><td>-</td>
 </tr>
 <tr>
-<td>BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)</td>
+<td>[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)</td>
 <td>-</td><td>1</td><td>-</td><td>-</td>
 </tr><tr>
-<td>BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)</td>
+<td>[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)</td>
 <td>-</td><td>-</td><td>1</td><td>-</td>
 </tr><tr>
-<td>BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)</td>
+<td>[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)</td>
 <td>-</td><td>-</td><td>-</td><td>1</td>
 </tr>
@@ Line 87: / Line 87: @@
 </table>
 :From left;
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> ?
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> ?
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
-::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::#[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
-::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> None?
+::#[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> None?
 |}
 {|align="justify"
@@ Line 123: / Line 123: @@
 From the left;
 （single digestuion）
-::5, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
+::5, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
-::6, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::6, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
-::7, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
+::7, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
-::8, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
+::8, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK
 |}

Team:Chiba/jk/β/week 2

From 2008.igem.org

Revision as of 11:52, 23 October 2008

Contents

Week 2

>last week

24 August, 2008

25 August, 2008

26 August, 2008

27 August, 2008

28 August, 2008

29 August, 2008

30 August, 2008

>next week