Team:Chiba/jk/β/week 3

From 2008.igem.org

(Difference between revisions)

Revision as of 08:33, 26 September 2008

>送受信班

Week 3

>last week

31 August, 2008

Transformation

Competent Cells : XL10G

Plac+RBS+LasI
Plac+RBS+RhlI

---> We saved these plates with a refrigerator.

Digestion

BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)

Sample No	1~3	4
Sample DNA	12	10
PstⅠ	0.2	0.2
XbaⅠ	0.2	-
SpeⅠ	-	0.4
Buffer 2	-	1.5
Buffer 3	2	-
BSA	2	1.5
dH2O	3.6	1.4
TOTAL	20	15

--->(1/9)Gel Check

(30/8)--->Mini prep

--->Digestion test

Plac+RBS+RhlI Sample No.2~5
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②

Sample	Single Digesiton 2~5	Double Digestion 2~5	Single Digestion T9002①	Single Digesiton T9002②	Double Digestion T9002①②
Sample DNA	1	3	3	1	3
XbaⅠ	0.1	0.1	0.1	0.1	0.1
SpeⅠ	-	0.1	-	-	0.1
Buffer 2	0.9	0.8	0.9	0.9	0.8
BSA	1	1	1	1	1
dH2O	7	5	5	7	5
TOTAL	10	10	10	10	10

--->Gel Check

Sample No	Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①②	Single & Double Digestion
Sample DNA	1	10
Loading Dye	1	2
dH2O	4	-
TOTAL	6	12

From Left

Single Digestion Plac+RBS+RhlI 2~5
Single Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
Double Digestion Plac+RBS+RhlI 2~5
Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②

1 September, 2008

(31/8)--->Gel Check

Sample No	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24	18

From left;

insert-1(I9026)

insert-2(I9030)

insert-3(S03154)

vector-4(R0010)

--->Gel extract

--->zymo

insert-1(I9026) -> 7μL

insert-2(I9030) -> 7μL

insert-3(S03154) -> 7μL

vector-4(R0010) -> 15μL

--->SAP

vector-4(R0010)

--->Zymo

vector-4(R0010) -> 20μL

--->Gel Check

Sample No	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24	18

From left;

insert-1(I9026) -> OK
insert-2(I9030) -> OK
insert-3(S03154) -> None
vector-4(R0010) -> OK

--->Ligation

Sample No	(1)	(2)	(3)	(4)	(5)
insert-1(I9026)	3	-	3	-	-
insert-2(I9030)	-	3	-	3	-
vector-4(R0010)	3	3	-	-	3
ligase	1	1	1	1	1
Buffer	1	1	1	1	1
dH2O	2	2	5	5	5
TOTAL	10	10	10	10	10

--->Transformation

Competent cells : XL10GOLD 30μL

BBa_K084009(Plac+RBS+RhlI+LVA)[http://partsregistry.org/Part:BBa_K084009] : Sample No.L1~L4
BBa_K084010(Plac+RBS+CinI+LVA)[http://partsregistry.org/Part:BBa_K084010] : Sample No.R1~R4

--->(2/9)OD

2 September, 2008

(31/8)--->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 left;

I9026

I9030

S03154

(1/9)--->Colony-PCR

Colony PCR of 8 colonies from ligation plates (1/9-(1),(2)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).

DNA Template	1
dNTP mix	5
Foward Primer	0.3
Reverse Primer	0.3
DNA polymerase TAQ	0.5
Thermopol Buffer	3
dH2O	20.5
TOTAL	30μL

95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃

--->Gel Check

Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

Plac+RBS+RhlI+LVA

R1 -> OK

R2 -> Bad

R3~R7 -> OK

R8 -> Bad

From left;

Plac+RBS+CinI+LVA

C1,C2 -> OK

C3 -> Bad

C4~C6 -> OK

From left;

Plac+RBS+CinI+LVA

C7,C8 -> OK

BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK

--->(3/9)Mini prep

(1/9)--->OD測

Transformation

Competent cells : XL10G 30μL

C0161(2007) [http://partsregistry.org/Part:BBa_C0161]
C0161(2006) [http://partsregistry.org/Part:BBa_C0161]
C0261(2007) [http://partsregistry.org/Part:BBa_C0261]
C0261(2006) [http://partsregistry.org/Part:BBa_C0261]

--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep

--->Gel Check

Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

S03154 -> OK
R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK

From left;

C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK

--->Digestion test

R1,R3~R7 (Plac+RBS+RhlI+LVA)
C1,C2,C4~C8 (Plac+RBS+CinI+LVA)

Digestion	Single	Double
Sample DNA	1	3
XbaⅠ	0.1	0.1
PstⅠ	0.1	0.1
Buffer 2	0.9	-
Buffer 3	-	-0.8
BSA	1	1
dH2O	7	5
TOTAL	10	10

--->Gel Check

Sample DNA	10
Loading Dye	2
TOTAL	12

From left;

Single Digestion : R1,R3~R7 -> OK
Single Digestion : C1,C2,C4~C8 -> OK

From left;

Double Digestion : R1,R3~R7 -> OK
Double Digestion : C1,C2,C4~C8 -> OK

(2/9)--->Phenotype-test : mix

Sample No.	1	2	3	4	5	6	7	8	9	10	11
L1	2	-	-	-	-	-	-	-	-	-	-
L2	-	2	-	-	-	-	-	-	-	-	-
L3	-	-	2	-	-	-	-	-	-	-	-
L4	-	-	-	2	-	-	-	-	-	-	-
R1	-	-	-	-	1	-	-	-	-	-	-
R2	-	-	-	-	-	1	-	-	-	-	-
R3	-	-	-	-	-	-	1	-	-	-	-
R4	-	-	-	-	-	-	-	1	-	-	-
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]	-	-	-	-	-	-	-	-	2	-	-
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]	2	2	2	2	1	1	1	1	2	1	1
AHL	1	1	1	1	1	1	1	1	1	1	1

|

| 8h

V

Spindown

|

V

the intensity GFP

4 September, 2008

--->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 left;

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 left;

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 left;

5 September, 2008

6 September, 2008

>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 187: / Line 187: @@
 :--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo]]'''
 ::vector-4(R0010) -> 20μL
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0901.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1~3</td><td>4</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>20</td><td>15</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>4</td><td>3</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>24</td><td>18</td>
+</tr>
+</table>
+:From left;
+::*insert-1(I9026) -> OK
+::*insert-2(I9030) -> OK
+::*insert-3(S03154) -> None
+::*vector-4(R0010) -> OK
+|}
@@ Line 196: / Line 225: @@
 </tr>
 <tr>
-<td>insert①C0178</td>
+<td>insert-1(I9026)</td>
-<td>1</td><td>-</td><td>-</td><td>1</td><td>-</td>
+<td>3</td><td>-</td><td>3</td><td>-</td><td>-</td>
 </tr>
 <tr>
-<td>insert②C0170</td>
+<td>insert-2(I9030)</td>
-<td>-</td><td>1</td><td>-</td><td>-</td><td>1</td>
+<td>-</td><td>3</td><td>-</td><td>3</td><td>-</td>
+</tr>
+<tr>
+<td>vector-4(R0010)</td>
+<td>3</td><td>3</td><td>-</td><td>-</td><td>3</td>
 </tr>
 <tr>
-<td>vector③J04500</td>
-<td>-</td><td>-</td><td>2</td><td>2</td><td>2</td>
-</tr>]
 <td>ligase</td>
 <td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
@@ Line 212: / Line 242: @@
 <tr>
 <td>Buffer</td>
-<td>2</td><td>2</td><td>2</td><td>2</td><td>2</td>
+<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 </tr>
 <tr>
 <td>dH2O</td>
-<td>6</td><td>6</td><td>5</td><td>4</td><td>4</td>
+<td>2</td><td>2</td><td>5</td><td>5</td><td>5</td>
 </tr>
 <tr>
@@ Line 225: / Line 255: @@
-'''[[Team:Chiba/protocol/transformation|Transformation]]'''
+:--->'''[[Team:Chiba/protocol/transformation|Transformation]]'''
-:Competent cells : BW ΔFliC  40μL
+:Competent cells : XL10GOLD  30μL
-::*BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007] : Sample No.L1~L4
+::*BBa_K084009(Plac+RBS+RhlI+LVA)[http://partsregistry.org/Part:BBa_K084009] : Sample No.L1~L4
-::*BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008] : Sample No.R1~R4
+::*BBa_K084010(Plac+RBS+CinI+LVA)[http://partsregistry.org/Part:BBa_K084010] : Sample No.R1~R4
 :--->(2/9)OD

Team:Chiba/jk/β/week 3

From 2008.igem.org

Revision as of 08:33, 26 September 2008

Contents

Week 3

31 August, 2008

1 September, 2008

2 September, 2008

3 September, 2008

4 September, 2008

5 September, 2008

6 September, 2008

>next week