Team:Chiba/jk/β/weak 2

From 2008.igem.org

(Difference between revisions)

Revision as of 07:17, 20 September 2008

>送受信班

Week 2

24 August, 2008

(22/8)--->Mini prep

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

--->Digestion test

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

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

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

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

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

25 August, 2008

(24/8)--->Digestion

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

--->Digestion (Double Digestion)

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

Insert:609bp -> OK

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

Insert:609bp -> OK

3, BBa_J04500[http://partsregistry.org/Part: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: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[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??

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 1: / Line 1: @@
 >[[Team:Chiba/jk/β|送受信班]]
+==Week 2==
+===24 August, 2008===
+:(22/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
+:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>5</td><td>6</td><td>7</td><td>8</td>
+</tr>
+<tr>
+<td>BBa_J04500[http://partsregistry.org/Part: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>-</td><td>1</td><td>-</td><td>-</td>
+</tr><tr>
+<td>BBa_J13002[http://partsregistry.org/Part: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>-</td><td>-</td><td>-</td><td>1</td>
+</tr>
+<tr>
+<td>EcoRⅠ</td>
+<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
+</tr>
+<tr>
+<td>Buffer 4</td>
+<td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>8</td><td>8</td><td>8</td><td>8</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>10</td><td>10</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0824-1.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1~8</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>4</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td>
+</tr>
+</table>
+:From left;
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> ?
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
+::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> None?
+|}
+{|align="justify"
+|[[Image:Chiba-0824-2.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>5~8</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>9</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td></tr>
+<tr>
+<td>dH2O</td>
+<td>1</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td>
+</tr>
+</table>
+From the left;
+（single digestuion）
+::5, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
+::6, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
+::7, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
+::8, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
+|}
+===25 August, 2008===
+:(24/8)--->'''[[Team:Chiba/protocol/digestion|Digestion]]'''
+::#Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::#Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
+::#Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
+::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1</td><td>2</td><td>3</td><td>4</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>5</td><td>5</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>EcoRⅠ</td>
+<td>0.5</td><td>o.5</td><td>-</td><td>-</td>
+</tr>
+<tr>
+<td>SpeⅠ</td>
+<td>0.5</td><td>0.5</td><td>-</td><td>-</td>
+</tr>
+<tr>
+<td>Buffer 1</td>
+<td>1</td><td>1</td><td>-</td><td>-</td>
+</tr>
+<tr>
+<td>Buffer 3</td>
+<td>-</td><td>-</td><td>1</td><td>-</td>
+</tr>
+<tr>
+<td>BSA</td>
+<td>1</td><td>1</td><td>-</td><td>-</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>2</td><td>2</td><td>7.5</td><td>-</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>10</td><td>10</td><td>10</td><td>1</td>
+</tr>
+</table>
+:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
+{|align="justify"
+|[[Image:Chiba-0825-1.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>10</td><td>1</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td><td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>-</td><td>4</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td><td>6</td>
+</tr>
+</table>
+:From right;
+::#Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
+::#Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
+::#Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
+::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)
+|}
+--->[[Team:Chiba/protocol/digestion|Digestion]]
+(Double Digestion)
+::#BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
+::#BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
+::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1</td><td>2</td><td>3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>33ng/μl×60μl</td><td>33ng/μl×60μl</td><td>100ng/μl×20μl</td>
+</tr>
+<tr>
+<td>PstⅠ</td>
+<td>2</td><td>2</td><td>2</td>
+</tr>
+<tr>
+<td>XbaⅠ</td>
+<td>2</td><td>2</td><td>-</td>
+</tr>]
+<td>SpeⅠ</td>
+<td>-</td><td>-</td><td>2</td>
+</tr>
+<tr>
+<td>Buffer 2</td>
+<td>-</td><td>-</td><td>5</td>
+</tr>
+<tr>
+<td>Buffer 3</td>
+<td>10</td><td>10</td><td>-</td>
+</tr>
+<tr>
+<td>BSA</td>
+<td>10</td><td>10</td><td>5</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>16</td><td>16</td><td>16</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>100</td><td>100</td><td>52</td>
+</tr>
+</table>
+===26 August, 2008===
+:(8/25)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0825-2.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1~3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>30</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>6</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>36</td>
+</tr>
+</table>
+::1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
+:::Insert:609bp -> OK
+|}
+{|align="justify"
+|[[Image:Chiba-0825-3.JPG]]
+|
+::2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
+:::Insert:609bp -> OK
+|}
+{|align="justify"
+|[[Image:Chiba-0825-4.JPG]]
+|
+::3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
+::: -> OK
+|}
+--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
+:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
+:::#5μl
+:::#5μl
+:::#27μl
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0825-5.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1~3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>2</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td></tr>
+<tr>
+<td>H2O</td>
+<td>8</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td></tr>
+</table>
+:From left;
+::#insert:C0178 -> OK
+::#insert:C0170 -> OK?
+::#vector:J04500 -> OK
+|}
+:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>26</td>
+</tr>
+<tr>
+<td>SAP</td>
+<td>1</td></tr>
+<tr>
+<td>SAP Buffer</td>
+<td>3</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>30μL</td></tr>
+</table>
+:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
+:::Sample No.3->9μl
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0826.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No.3</td>
+<td>1</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>4</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td></tr>
+</table>
+::Sample No.3 -> OK ･･･30ng?
+|}
+:--->'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>(1)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
+</tr>
+<tr>
+<td>insert①C0178</td>
+<td>1</td><td>-</td><td>-</td><td>1</td><td>-</td>
+</tr>
+<tr>
+<td>insert②C0170</td>
+<td>-</td><td>1</td><td>-</td><td>-</td><td>1</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>
+</tr>
+<tr>
+<td>Buffer</td>
+<td>2</td><td>2</td><td>2</td><td>2</td><td>2</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>6</td><td>6</td><td>5</td><td>4</td><td>4</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/transformation|Transformation]]'''
+::#insert①C0178
+::#insert②C0170
+::#vector③J04500
+::#insert①C0178+vector③J04500
+::#insert②C0170+vector③J04500
+===27 August, 2008===
+[[Team:Chiba/protocol/digestion|Digestion Test]]
+::Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1</td><td>2</td><td>3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>PstⅠ</td>
+<td>-</td><td>-</td><td>0.5</td>
+</tr>
+<tr>
+<td>EcoRⅠ</td>
+<td>-</td><td>0.5</td><td>0.5</td>
+</tr>
+<tr>
+<td>Buffer 3</td>
+<td>-</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>BSA</td>
+<td>-</td><td>-</td><td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>-</td><td>7.5</td><td>2</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>1</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0827.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1</td><td>2</td><td>3</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>5</td><td>5</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>H2O</td>
+<td>4</td><td>-</td><td>-</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td><td>6</td><td>6</td>
+</tr>
+</table>
+:From left;<br>
+:#BBa_R0010
+:#Single Digestion
+:#Double Digestion
+|}
+:(26/8)--->'''[[Team:Chiba/protocol/PCR|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)).
+:<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>5</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>0.5</td>
+</tr>
+<tr>
+<td>Thermopol Buffer</td>
+<td>5</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>28.5</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>50μL</td>
+</tr>
+</table>
+:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0827-2.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample DNA</td>
+<td>10</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td></tr>
+</table>
+:From left;<br>
+:1~5  insert:C0170 + vector:J04500  -> OK
+:6    F2620･･Positive Control  -> OK
+:7~11 insert:C0178 + vector:J04500 -> OK
+|}
+:--->(28/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Miniprep]]'''
+'''[[Team:Chiba/protocol/transformation|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]
+:--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
+:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+===28 August, 2008===
+:(27/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
+::#insert:C0170 + vector:J04500
+::#insert:C0178 + vector:J04500
+:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
+::#insert:C0170 + vector:J04500
+::#insert:C0178 + vector:J04500
+<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>3</td><td>4</td><td>5</td><td>6</td>
+</tr>
+<tr>
+<td>insert:C0170 + vector:J04500 </td>
+<td>1</td><td>-</td><td>1</td><td>-</td>
+</tr>
+<tr>
+<td>insert:C0178 + vector:J04500 </td>
+<td>-</td><td>1</td><td>-</td><td>1</td>
+</tr>
+<tr>
+<td>EcoRⅠ</td>
+<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
+</tr>
+<tr>
+<td>SpeⅠ</td>
+<td>-</td><td>-</td><td>0.1</td><td>0.1</td>
+</tr>
+<tr>
+<td>Buffer 3</td>
+<td>1</td><td>1</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>BSA</td>
+<td>-</td><td>-</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>7.8</td><td>7.8</td><td>6.8</td><td>6.8</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>10</td><td>10</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0828.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1,2</td><td>3~6</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>10</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td><td>2</td>
+</tr>
+<tr>
+<td>H2O</td>
+<td>4</td><td>-</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td><td>12</td>
+</tr>
+</table>
+:From left;
+::#　-> OK
+::#　-> OK
+::#　-> OK
+::#　-> OK
+::#　-> OK
+::#　-> OK
+|}
+===29 August, 2008===
+:--->'''[[Team:Chiba/protocol/digestion|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]
+<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>S1~10</td><td>W1</td><td>W2~10</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>4</td><td>2</td>
+</tr>
+<tr>
+<td>PstⅠ</td>
+<td>-</td><td>0.075</td><td>0.1</td>
+</tr>
+<tr>
+<td>EcoRⅠ</td>
+<td>0.1</td><td>0.075</td><td>0.1</td>
+</tr>
+<tr>
+<td>Buffer 3</td>
+<td>1</td><td>0.75</td><td>1</td>
+</tr>
+<tr>
+<td>BSA</td>
+<td>-</td><td>0.75</td><td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>7.9</td><td>4.35</td><td>5.8</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>10</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0829.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No.</td>
+<td>1~10</td><td>S1~S10,W1~W10</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>10</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td><td>2</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td><td>12</td></tr>
+</table>
+:From left;<br>
+::1~5,S1,S5,W1~W5
+|}
+{|align="justify"
+|[[Image:Chiba-0829-2.JPG]]
+|
+:From left;<br>
+::6~10,S6~S10,W6~W10
+|}
+:(27/8)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0829-3.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample DNA</td>
+<td>0.5</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>4.5</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>6μL</td></tr>
+</table>
+::R0010 -> OK
+|}
+'''[[Team:Chiba/protocol/digestion|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]
+<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>100ng/μL×20</td><td>200ng/μL×30</td>
+</tr>
+<tr>
+<td>PstⅠ</td>
+<td>2</td><td>2</td>
+</tr>
+<tr>
+<td>XbaⅠ</td>
+<td>2</td><td>-</td>
+</tr>
+<tr>
+<td>SpeⅠ</td>
+<td>-</td><td>2</td></tr>
+<tr>
+<td>Buffer 2</td>
+<td>-</td><td>5</td></tr>
+<tr>
+<tr>
+<td>Buffer 3</td>
+<td>5</td><td>-</td></tr>
+<tr>
+<td>BSA</td>
+<td>5</td><td>5</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>16</td><td>6</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>50</td><td>50</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0829-4.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>10</td><td>3</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>2</td><td>2</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>-</td><td>7</td>
+</tr>
+<tr>
+<td>TOTAL</td>
+<td>12</td><td>12</td></tr>
+</table>
+:insert-I9026 : 685bp -> None<br>
+|}
+{|align="justify"
+|[[Image:Chiba-0829-5.JPG]]
+|
+:insert-I9030 : 745bp -> None<br>
+|}
+{|align="justify"
+|[[Image:Chiba-0829-6.JPG]]
+|
+:insert-S03154 : 685bp -> None<br>
+|}
+{|align="justify"
+|[[Image:Chiba-0829-7.JPG]]
+|
+:vector-R0010 : 2079bp -> OK<br>
+|}
+:--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
+:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
+:::->15μL
+:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>4</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>15</td>
+</tr>
+<tr>
+<td>SAP</td>
+<td>2</td></tr>
+<tr>
+<td>SAP Buffer</td>
+<td>3</td></tr>
+<tr>
+<td>dH2O</td>
+<td>10</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>30μL</td></tr>
+</table>
+:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|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===
+:'''[[Team:Chiba/protocol/transformation|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)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
+:(29/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
+::#insert:C0170 + vector:J04500
+::#insert:C0178 + vector:J04500
+:--->'''[[Team:Chiba/protocol/digestion|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
+:<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="270">Sample No.</td><td>Single : 10~18</td><td>Double : 19~27</td>
+</tr>
+<tr>
+<td>Sample DNA</td><td>1</td><td>5</td>
+</tr>
+<tr>
+<td>XbaⅠ</td><td>0.1</td><td>0.1</td>
+</tr>
+<tr>
+<td>SpeⅠ</td><td>-</td><td>0.1</td>
+</tr>
+<tr>
+<td>Buffer 2</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>BSA</td><td>1</td><td>1</td>
+</tr>
+<tr>
+<td>dH2O</td><td>6.9</td><td>2.8</td>
+</tr>
+<tr>
+<td>TOTAL</td><td>10</td><td>10</td>
+</tr>
+</table>
+:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+{|align="justify"
+|[[Image:Chiba-0830.JPG]]
+|
+:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+<tr>
+<td width="257">Sample No</td>
+<td>1~9</td><td>10~18</td><td>19~27</td>
+</tr>
+<tr>
+<td>Sample DNA</td>
+<td>1</td><td>10</td><td>10</td>
+</tr>
+<tr>
+<td>Loading Dye</td>
+<td>1</td><td>2</td><td>2</td>
+</tr>
+<tr>
+<td>dH2O</td>
+<td>4</td><td>-</td><td>-</td></tr>
+<tr>
+<td>TOTAL</td>
+<td>6</td><td>12</td><td>12</td>
+</tr>
+</table>
+:From left;<br>
+::Sanple No.1~13
+::-> OK
+|}
+{|align="justify"
+|[[Image:Chiba-0830-2.JPG]]
+|
+:From left;<br>
+::Sample No.14~17,24~16
+::14 -> OK
+::15,16 -> Bad
+::17 -> None
+::24~16 -> Bad
+|}
+{|align="justify"
+|[[Image:Chiba-0830-3.JPG]]
+|
+:From left;<br>
+::Sample No.18,27,19~23
+::18,27 -> Bad
+::19~22 -> OK
+::23 -> OK??
+|}
 {| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"

Team:Chiba/jk/β/weak 2

From 2008.igem.org

Revision as of 07:17, 20 September 2008

Contents

Week 2

24 August, 2008

25 August, 2008

26 August, 2008

27 August, 2008

28 August, 2008

29 August, 2008

30 August, 2008