Team:Chiba/jk/β/week 2

From 2008.igem.org

(Difference between revisions)

Revision as of 12:01, 23 October 2008

>送受信班

>Protocols

Week 2

>last week

24 August, 2008

(22/8)--->Mini prep

[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006)

--->Digestion test

[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006)

Sample No.	5	6	7	8
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)	1	-	-	-
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)	-	1	-	-
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)	-	-	1	-
[http://partsregistry.org/Part:BBa_J13002 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;

[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> ?
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> None?

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

From the left; （single digestuion）

5, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK

6, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK

7, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK

8, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK

25 August, 2008

(24/8)--->Digestion

Double Digestion:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
Double Digesiton:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
Single Digestion:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](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:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
Double Digesiton:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> ?(low)
Single Digestion:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> ?(low)
[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> OK(low)

--->Digestion (Double Digestion)

[http://partsregistry.org/Part:BBa_C0178 BBa_C0178]
[http://partsregistry.org/Part:BBa_C0170 BBa_C0170]
[http://partsregistry.org/Part:BBa_J04500 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
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, [http://partsregistry.org/Part:BBa_C0178 BBa_C0178]

Insert:609bp -> OK

2, [http://partsregistry.org/Part:BBa_C0170 BBa_C0170]

Insert:609bp -> OK

3, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](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:[http://partsregistry.org/Part:BBa_C0178] --> OK
insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] --> OK?
vector:[http://partsregistry.org/Part:BBa_J04500 BBa_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

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[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

(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

--->(28/8)Miniprep

Transformation

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

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;

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) [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;

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 281: / Line 281: @@
 </tr>
 </table>
-::1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
-:::Insert:609bp -> OK
+:1, [http://partsregistry.org/Part:BBa_C0178 BBa_C0178]
+::Insert:609bp -> OK
 |}
 {|align="justify"
 |[[Image:Chiba-0825-3.JPG]]
 |
-::2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
+::2, [http://partsregistry.org/Part:BBa_C0170 BBa_C0170]
 :::Insert:609bp -> OK
 |}
@@ Line 293: / Line 295: @@
 |[[Image:Chiba-0825-4.JPG]]
 |
-::3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::3, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
-::: -> OK
+::: --> OK
 |}
@@ Line 332: / Line 334: @@
 :From left;
-::#insert:C0178 -> OK
+::#insert:[http://partsregistry.org/Part:BBa_C0178] --> OK
-::#insert:C0170 -> OK?
+::#insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] --> OK?
-::#vector:J04500 -> OK
+::#vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] --> OK
 |}
@@ Line 362: / Line 364: @@
 :--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
-:::Sample No.3->9μl
+:::Sample No.3 -->9μl
@@ Line 407: / Line 409: @@
 <td>vector③J04500</td>
 <td>-</td><td>-</td><td>2</td><td>2</td><td>2</td>
-</tr>]
+</tr>
+<tr>
 <td>ligase</td>
 <td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
@@ Line 432: / Line 435: @@
 ::#insert①C0178+vector③J04500
 ::#insert②C0170+vector③J04500
 ===27 August, 2008===

Team:Chiba/jk/β/week 2

From 2008.igem.org

Revision as of 12:01, 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