"

 

From 2008.igem.org

Revision as of 08:27, 9 September 2008 (view source) Yoshimi (Talk | contribs) ← Older edit		Revision as of 08:41, 9 September 2008 (view source) Yoshimi (Talk | contribs) (→Week 2) Newer edit →
Line 758:		Line 758:
	|		|
	From left;<br>		From left;<br>
-	:1~5  insert:C0170 + vector:J04500	+	:1~5  insert:C0170 + vector:J04500 -> OK
-	:6    F2620･･Positive Control	+	:6    F2620･･Positive Control -> OK
-	:7~11 insert:C0178 + vector:J04500	+	:7~11 insert:C0178 + vector:J04500 -> OK
	|}		|}
-	--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
-	--->(28/8)[[Team:Chiba/protocol/digestion|Digestion Test]]
		+	:--->(28/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''[[Team:Chiba/protocol/digestion|Digestion Test]]
-	:'''[[Team:Chiba/protocol/transformation|Transformation]]'''	+
		+	'''[[Team:Chiba/protocol/transformation|Transformation]]'''
	comptent cell : XL10G		comptent cell : XL10G
	::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]		::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
Line 782:		Line 782:
	::*BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]		::*BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]
-	--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''	+	:--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
	:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''		:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
Line 788:		Line 788:
	*28 August, 2008]		*28 August, 2008]
		+	(27/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
		+	::①insert:C0170 + vector:J04500
		+	::②insert:C0178 + vector:J04500
		+
		+
		+	:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
		+
		+	:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
		+	<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
		+	<tr>
		+	<td width="257">Sample No</td>
		+	<td>①②</td><td>Single Digesiton:①②</td><td>Double Digesiton:①②</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>H2O</td>
		+	<td>4</td><td>-</td><td>-</td>
		+	</tr>
		+	<tr>
		+	<td>TOTAL</td>
		+	<td>6</td><td>12</td><td>12</td>
		+	</tr>
		+	</table>
	{|align="justify"		{|align="justify"
	|[[Image:Chiba-0828.JPG]]		|[[Image:Chiba-0828.JPG]]
	|		|
-	From left;	+	From left;<br>
		+	:①
		+	:②
		+	:Single Digestion:①
		+	:Single Digestion:②
		+	:Double Digestion:①
		+	:Double Digestion:②
	|}		|}

---

Revision as of 08:41, 9 September 2008

β班の実験ノート

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4

Week 1

• 17 August, 2008

Transformation

• BBa_R0040(2008) [http://partsregistry.org/Part:BBa_R0040] -> No colonies on plates
• BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] -> No colonies on plates
• BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
• BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
• BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
• BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] -> No colonies on plates

• 18 August, 2008

Transformation

• BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
• BBa_C0076(2006) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
• BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
• BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
• BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
• BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]

--->(20/8)Mini prep

• 19 August, 2008

PCR

• BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
• BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
• BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
• BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
• BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154]
• BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154]
• BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control

--->Gel Check

BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] ->None BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] ->None BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] ->None BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] ->None BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154] ->None BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154] ->None BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control ->OK

• 20 August, 2008

(18/8)--->Mini prep

• BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
• BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
• BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
• BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
• BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]

--->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 the left; ①BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK ②BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK ③BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK ④BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK

BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK

BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None

BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> OK BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK

--->Digestion

• BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]

• 21 August, 2008

(20/8)--->PCR

• BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
• BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
• BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
• BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]

--->Gel Check

From the left; BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK

Transformation

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

• 22 August, 2008

Transformation

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

(Locate & 2 Punch ~iyama ver.~)

• BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
• BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
• BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates

• 23 August, 2008

--->Mini prep

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

--->Digestion

• BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
• BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
• BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
• BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
• BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
• BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
• BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]

--->Gel Check</table>

From the left; ①BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK ②BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK ③BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK ④BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK

BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]

BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] M BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]

BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Single Digestion BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Double Digestion

Week 2

• 24 August, 2008

(22/8)--->Mini prep

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

--->Digestion

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

--->Gel Check

Sample No	①	②	③	④	⑤	⑥	⑦	⑧
Sample DNA	1	1	1	1	1	1	1	1
Loading Dye	1	1	1	1	1	1	1	1
dH2O	4	4	4	4	4	4	4	4
TOTAL	6	6	6	6	6	6	6	6

From the left; ⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) ⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) ⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) ⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) （↓single digestuion） ⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) ⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) ⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) ⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)

Sample No	⑤	⑥	⑦	⑧
Sample DNA	9	9	9	9
Loading Dye	2	2	2	2
dH2O	1	1	1	1
TOTAL	12	12	12	12

From the left; （single digestuion） ⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) ⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) ⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) ⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)

• 25 August, 2008

(24/8)--->Digestion

①Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)

②Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)

③Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)

④BBa_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	②	③	④
Sample DNA	10	10	10	10
Loading Dye	2	2	2	1
dH2O	-	-	-	4
TOTAL	12	12	12	6

From right; ①Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK ②Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low) ③Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low) ④BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)

--->Digestion

①BBa_C0178[http://partsregistry.org/Part:BBa_C0178]

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

③BBa_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

--->Gel Check

Sample No	①	②	③
Sample DNA	30	30	30
Loading Dye	6	6	6
TOTAL	36	36	36

①BBa_C0178[http://partsregistry.org/Part:BBa_C0178] Insert:609bp -> OK

②BBa_C0170[http://partsregistry.org/Part:BBa_C0170] Insert:609bp -> OK

③BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK

• 26 August, 2008

(8/25)--->Gel Extract

--->Zymo Clean

①5μl

②5μl

③27μl

--->Gel Check

Sample No	①	②	③
Sample DNA	2	2	2
Loading Dye	2	2	2
H2O	8	8	8
TOTAL	12	12	12

From left; ①insert:C0178 -> OK ②insert:C0170 -> OK? ③Vector:J04500 -> OK

--->Dephosphorylation

③

--->Zymo Clean

③->9μl

--->Gel Check

Sample DNA ③	1
Loading Dye	1
dH2O	4
TOTAL	6

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

(1)insert①C0178

(2)insert②C0170

(3)vector③J04500

(4)insert①C0178+vector③J04500

(5)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; ①BBa_R0010 ②Single Digestion ③Double Digestion

(26/8)--->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)).

]

</tr>

Sample No	(4)-1~5	(5)-1~5	(F2620)
insert①C0178	1	-	-
insert②C0170	-	1	-
vector③J04500	-	-	2
ligase	1	1	1
Buffer	2	2	2
dH2O	6	6	5
TOTAL	10	10	10

--->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)Mini prepDigestion Test

Transformation comptent cell : XL10G

• BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
• BBa_C0062(2006) [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_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]

--->Mini prep

--->(29/8)Gel Check

• 28 August, 2008]

(27/8)--->Mini prep

①insert:C0170 + vector:J04500

②insert:C0178 + vector:J04500

--->Digestion Test

--->(29/8)Gel Check

Sample No	①②	Single Digesiton:①②	Double Digesiton:①②
Sample DNA	1	10	10
Loading Dye	1	2	2
H2O	4	-	-
TOTAL	6	12	12

From left; ① ② Single Digestion:① Single Digestion:② Double Digestion:① Double Digestion:②

• 29 August, 2008
• 30 August, 2008

Week 3

• 31 August, 2008

• 1 September, 2008

• 2 September, 2008

• 3 September, 2008

• 4 September, 2008

• 5 September, 2008

• 6 September, 2008

Week 4

• 7 September, 2008

• 8 September, 2008

• 9 September, 2008

• 10 September, 2008

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