Team:Chiba/Calendar-Home/30 August 2008

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(Difference between revisions)

Revision as of 11:03, 24 October 2008

>Home | Notebook

29 August 2008 <|> 31 August 2008

Laboratory work

Team:Input

Digestion

x-RBS-cI-s-p(780bp)

混ぜ表

	double
dH₂O(μL)	6
10×BSA(μL)	10
10×NE(μL)	10
PstI(μL)	2
XbaI(μL)	2
DNA(μL)	33.6ng/μl×30μl(1μg)
TOTAL(μL)	60

-->30μlゲル抽出

-->ゲル抽出

-->Zymo Clean

5μ抽出,うち1μlをゲルチェック-->OK,4μlはLigation用

Digestion

-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)

混ぜ表

	double
dH₂O(μL)	4
10×BSA(μL)	10
10×NE(μL)	10
PstI(μL)	2
SpeI(μL)	4
DNA(μL)	75ng/μl×30μl(2μg)
TOTAL(μL)	60

-->30μlゲル抽出

ゲル抽出

-->Zymo Clean

-->SAP

混ぜ表


dH₂O(μL)	9
DNA(μL)	10
SAP(μL)	1
SAP Buffer(μL)	10
TOTAL(μL)	30

5μ抽出,うち1μlをゲルチェック

-->OK,4μlはLigation用

Ligation

混ぜ表

	-	b.g
dH₂O(μL)	1.4	8
Ligase(μL)	1	1
Ligase Buffer(μL)	2	2
Vector(μL)	1.8	1
Insert(μL)	3	-
TOTAL(μL)	10	10

-->Transformation(XL10G)

-->CFU40(b.gは22)

コロニーPCR(16コつつく):PCR

混ぜ表


VF(μL)	2
VR2(μL)	2
dNTP(μL)	2
thermo pol buffer(μL)	2
Tag(μL)	0.3
dH₂O(μL)	11.7
TOTAL(μL)	20

-->Insertチェックしたら1つだけ正しい位置にバンドが出現。そのコロニーを2ml培養し、mini prepした。

-->Mini prep

Digestion

x-RBS-cI-s-p(780bp)

混ぜ表

	double
dH₂O(μL)	6
10×BSA(μL)	10
10×NE(μL)	10
PstI(μL)	2
XbaI(μL)	2
DNA(μL)	33.6ng/μl×30μl(1μg)
TOTAL(μL)	60

Gel Extract

混ぜ表


Digested DNA Sample(μL)	30
Loading Dye(μL)	6
TOTAL(μL)	36

-->Zymo Clean

-->NFW5μlで溶出

-->Gel Check

混ぜ表


dH₂O(μL)	4
DNA(μL)	1
Loading Dye(μL)	1
TOTAL(μL)	6

-->Gel Check-->OK

Digestion

-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)

混ぜ表

	double
dH₂O(μL)	4
10×BSA(μL)	10
10×NE(μL)	10
PstI(μL)	2
SpeI(μL)	4
DNA(μL)	75ng/μl×30μl(2.25μg)
TOTAL(μL)	60

Gel Extract

混ぜ表


Digested DNA Sample(μL)	30
Loading Dye(μL)	6
TOTAL(μL)	36

-->Zymo Clean

-->NFW10μlで溶出

-->SAP

混ぜ表


dH₂O(μL)	9
DNA(μL)	10
SAP(μL)	1
SAP Buffer(×10)(μL)	10
TOTAL(μL)	30

-->37℃で1h、乾燥機(65℃)で15min,

-->Binding Bufferを90μl加え、VORTEX -->Zymo Clean

-->NFW5μlで抽出

Gel Check

混ぜ表


dH₂O(μL)	4
DNA(μL)	1
Loading Dye(μL)	1
TOTAL(μL)	6

-->ゲルチェックOK

Ligation

混ぜ表

	-	b.g
dH₂O(μL)	1.4	8
Ligase(μL)	1	1
Ligase Buffer(μL)	2	2
Vector(μL)	1.8	1
Insert(μL)	3	-
TOTAL(μL)	10	10

-->left at rest at room temparature(25℃?本当に?) -->Transformation(XL10G):

-->CFU40(b.gは22)

Team:Communication

Transformation

competent cells : XL10G

[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)
[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
[http://partsregistry.org/Part:BBa_S03156 BBa_S03156](2007)
[http://partsregistry.org/Part:BBa_S03158 BBa_S03158](2007)
[http://partsregistry.org/Part:BBa_S03160 BBa_S03160](2007)
[http://partsregistry.org/Part:BBa_C0062 BBa_C0062](2007)
[http://partsregistry.org/Part:BBa_C0179 BBa_C0179](2007)

--->(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
[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007) -> 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??

Team:Output

Transformation

mcherry

PCR

[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](rLuc)

Sample No.	[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
DNA tamplate	1
rLuc_fwd	2.5
Rev primer	2.5
Thermo pol Buffer	5
dNTPmix	5
Vent pol	0.5
dH₂O	34
TOTAL	50

@@ Line 14: / Line 14: @@
 <td>double</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>6</td>
 </tr>
 <tr>
-<td>10×BSA</td>
+<td>10×BSA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>10×NE</td>
+<td>10×NE(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>PstI</td>
+<td>PstI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>XbaI</td>
+<td>XbaI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>33.6ng/μl×30μl(1μg)</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>60</td>
 </tr>
@@ Line 61: / Line 61: @@
 <td>double</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>10×BSA</td>
+<td>10×BSA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>10×NE</td>
+<td>10×NE(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>PstI</td>
+<td>PstI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>SpeI</td>
+<td>SpeI(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>75ng/μl×30μl(2μg)</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>60</td>
 </tr>
@@ Line 103: / Line 103: @@
 <td></td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>9</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>SAP</td>
+<td>SAP(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>SAP Buffer</td>
+<td>SAP Buffer(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>30</td>
 </tr>
@@ Line 135: / Line 135: @@
 <td>-</td><td>b.g</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>1.4</td><td>8</td>
 </tr>
 <tr>
-<td>Ligase</td>
+<td>Ligase(μL)</td>
 <td>1</td><td>1</td>
 </tr>
 <tr>
-<td>Ligase Buffer</td>
+<td>Ligase Buffer(μL)</td>
 <td>2</td><td>2</td>
 </tr>
 <tr>
-<td>Vector</td>
+<td>Vector(μL)</td>
 <td>1.8</td><td>1</td>
 </tr>
 <tr>
-<td>Insert</td>
+<td>Insert(μL)</td>
 <td>3</td><td>-</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>10</td><td>10</td>
 </tr>
@@ Line 174: / Line 174: @@
 </tr>
 <tr>
-<td>VF</td>
+<td>VF(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>VFR</td>
+<td>VR2(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>dNTP</td>
+<td>dNTP(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>thermo pol buffer</td>
+<td>thermo pol buffer(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>Tag</td>
+<td>Tag(μL)</td>
 <td>0.3</td>
 </tr>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>11.7</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>20</td>
 </tr>
@@ Line 219: / Line 219: @@
 <td>double</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>6</td>
 </tr>
 <tr>
-<td>10×BSA</td>
+<td>10×BSA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>10×NE</td>
+<td>10×NE(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>PstI</td>
+<td>PstI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>XbaI</td>
+<td>XbaI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>33.6ng/μl×30μl(1μg)</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>60</td>
 </tr>
@@ Line 255: / Line 255: @@
 <td></td>
 <tr>
-<td>Digestion産物</td>
+<td>Digested DNA Sample(μL)</td>
 <td>30</td>
 </tr>
 <tr>
-<td>Dye</td>
+<td>Loading Dye(μL)</td>
 <td>6</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>36</td>
 </tr>
@@ Line 280: / Line 280: @@
 <td></td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>Dye</td>
+<td>Loading Dye(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>6</td>
 </tr>
@@ Line 309: / Line 309: @@
 <td>double</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>10×BSA</td>
+<td>10×BSA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>10×NE</td>
+<td>10×NE(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>PstI</td>
+<td>PstI(μL)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>SpeI</td>
+<td>SpeI(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>75ng/μl×30μl(2.25μg)</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>60</td>
 </tr>
@@ Line 345: / Line 345: @@
 <td></td>
 <tr>
-<td>Digestion産物</td>
+<td>Digested DNA Sample(μL)</td>
 <td>30</td>
 </tr>
 <tr>
-<td>Dye</td>
+<td>Loading Dye(μL)</td>
 <td>6</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>36</td>
 </tr>
@@ Line 371: / Line 371: @@
 <td></td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>9</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>SAP</td>
+<td>SAP(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>SAP Buffer(×10)</td>
+<td>SAP Buffer(×10)(μL)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>30</td>
 </tr>
@@ Line 406: / Line 406: @@
 <td></td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>4</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>Dye</td>
+<td>Loading Dye(μL)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>6</td>
 </tr>
@@ Line 431: / Line 431: @@
 <td>-</td><td>b.g</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>1.4</td><td>8</td>
 </tr>
 <tr>
-<td>Ligase</td>
+<td>Ligase(μL)</td>
 <td>1</td><td>1</td>
 </tr>
 <tr>
-<td>Ligase Buffer</td>
+<td>Ligase Buffer(μL)</td>
 <td>2</td><td>2</td>
 </tr>
 <tr>
-<td>Vector</td>
+<td>Vector(μL)</td>
 <td>1.8</td><td>1</td>
 </tr>
 <tr>
-<td>Insert</td>
+<td>Insert(μL)</td>
 <td>3</td><td>-</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>10</td><td>10</td>
 </tr>
 </table>
--->25℃で2h放置
+-->left at rest at room temparature(25℃?本当に?)
 -->'''[[Team:Chiba/protocol/transformation|Transformation]]'''(XL10G):
 -->CFU40(b.gは22)
 ===Team:Communication===

Team:Chiba/Calendar-Home/30 August 2008

From 2008.igem.org

Revision as of 11:03, 24 October 2008

Contents

Laboratory work

Team:Input

Team:Communication

Team:Output