Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

(Difference between revisions)

Latest revision as of 18:50, 25 September 2013

Sep 2

Colony PCR

Tatsui

Sample	base pair
9/1 RBS-lysis2-DT-(1)	985
Ptet-RBS-lacZα-DT-(1)	765
Plac-RBS-lacZα-DT-(1)	765
Plac-RBS-lacZα-DT-(2)	765

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	9/1 RBS-lysis2+DT -(1)
3	9/1 Ptet+RBS-lacZα-DT -(1)
4	9/1 Plac+RBS-lacZα-DT -(1)
5	9/1 Plac+RBS-lacZα-DT -(2)
6	100bp ladder

Restriction Enzyme Digestion

Nakamoto

	pSB1C3-(1)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts(EcoRI+SpeI)	7µL	1µL	1µL	0µL	0µL	3µL	0µL	0.3µL	17.7µL	30µL
NC(EcoRI+SpeI)	0.3µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.6µL	10µL
2 cuts(XbaI+PstI)	7µL	0µL	0µL	1µL	1µL	0µL	3µL	0.3µL	17.7µL	30µL
NC(XbaI+PstI)	0.3µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	8/21 tRMA-spinach(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	8µL	1.0µL	1.0µL	3µL	0.3µL	17.6µL	30µL
NC	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/21 tetR aptamer12_1R-(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	6µL	1.0µL	1.0µL	3µL	0.3µL	18.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	0.1µL	8.5µL	10µL

	8/21 pT181 attenuator-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts(EcoRI+SpeI)	8µL	1µL	1µL	0µL	0µL	3µL	0µL	0.3µL	16.3µL	30µL
NC(EcoRI+SpeI)	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.4µL	10µL
2 cuts(XbaI+PstI)	8µL	0µL	0µL	1µL	1µL	0µL	3µL	0.3µL	16.3µL	30µL
NC(XbaI+PstI)	0.5µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

Liquid Culture

Kojima

Sample	medium
tRNA-Spinach-1	Plusgrow medium(+CP)
tetR aptamaer12_1R-1	Plusgrow medium(+CP)
pT181 attenuator-1	Plusgrow medium(+CP)
pT181 antisense-1	Plusgrow medium(+CP)

Master Plate

Kojima

Number	Use LB plate(+CP)
1	tRNA Spinach-1
2	tetR aptamaer12_1R
3	tetR aptamaer12_P
4	tetR aptamaer12_1M
5	pT181 attenuator-2
6	Fusion1 attenuator-1
7	Fusion3m2 attenuator-1
8	pT181 antisense-1
9	Fusion1 antisense-1
10	Fuaion6 antisense-1

Electrophoresis

Nakamoto & Tatsui

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	pSB1C3 -(1)	EcoRI	SpeI
3	pSB1C3 -(1)	--	--
5	pSB1C3 -(1)	XbaI	PstI
6	pSB1C3 -(1)	--	--
7	pT181 attenuator (2)	EcoRI	SpeI
8	pT181 attenuator (2)	--	--
9	pT181 attenuator (2)	XbaI	SpeI
10	pT181 attenuator (2)	--	--
11	tetR-aptamer 12_1R	EcoRI	SpeI
12	tetR-aptamer 12_1R	--	--
13	tRNA Spinach (1)	EcoRI	SpeI
14	100bp ladder	--	--
15	tRNA Spinach (1)	--	--

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	-
2	pSB1C3(EcoRI+SpeI)	EcoRI&SpeI
3	pSB1C3(EcoRI+SpeI)	EcoRI&SpeI
4	--	--
5	8/24 tetR aptamer 12_1R	EcoRI&SpeI
6	8/24 tetR aptamer 12_1R	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder	-
2	pT181 attenuator(2)(EcoRI+SpeI)	EcoRI&SpeI
3	pT181 attenuator(2)(EcoRI+SpeI)	EcoRI&SpeI
4	--	--
5	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
6	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
7	--	--
8	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
9	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
10	--	--
11	Spinach	EcoRI&SpaI
12	Spinach	EcoRI&SpaI

Name	concentration[µg/mL]	260/280	260/230
pSB1C3 (EcoRI&SpeI)	3.0	2.18	0.31
pSB1C3 (XbaI&PstI)	4.7	2.25	0.36
pT181 attenuator-(2)(EcoR&SpeI)	8.6	2.74	0.01
pT181 attenuator-(2) (XbaI&PstI)	16.5	2.46	0.03
Spinach (EcoRI&SpeI)	2.8	2.98	0.27
tetR aptamer12_1R (EcoRI&SpeI)	50.4	28.07	1.97

Colony PCR

No name

Sample	base pair
9/1 Spinach(pSB1C3)	about 450
9/1 Spinach-DT	596
9/1 RBS-lysis1-DT	613

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Sample	base pair
9/1 Ptet-RBS-lacZα-DT-2	738
9/1 Ptet-RBS-lacZα-DT-3	738
9/1 Ptet-RBS-lacZα-DT-4	738
9/1 Ptet-RBS-lacZα-DT-5	738
9/1 Ptet-RBS-lacZα-DT-6	738
9/1 RBS-lysis2-DT-2	985
9/1 RBS-lysis2-DT-3	985

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Spinach(pSB1C3)
3	Spinach-DT
4	RBS-lysis1-DT
5	100bp ladder

Master Plate

No name

Number	Use LB plate(+CP)
1	9/1 Spinach(pSB1C3)
2	9/1 Spinach-DT-(1)

Liquid Culture

No name

Sample	medium
pT181 attenuator	Plusgrow medium (+CP)
pT181 antisense	Plusgrow medium (+CP)
Spinach	Plusgrow medium (+CP)

incubated at 37 °C for 1 hour

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
8/28 RBS-lysis3-DT	3µL	30µL	33µL	CP

@@ Line 22: / Line 22: @@
 |5min||30s||30s||1min||30cycles
 |}
-[[File:Igku Sep2 ColonyPCR (N1-2).jpg]]
+</div>
+===Electrophoresis===
+<div class="experiment">
+<span class="author">No name</span>
+{| class="wikitable"
+!Lane||Sample
+|-
+|1||100bp ladder
+|-
+|2||9/1 RBS-lysis2+DT -(1)
+|-
+|3||9/1 Ptet+RBS-lacZ&alpha;-DT -(1)
+|-
+|4||9/1 Plac+RBS-lacZ&alpha;-DT -(1)
+|-
+|5||9/1 Plac+RBS-lacZ&alpha;-DT -(2)
+|-
+|6||100bp ladder
+|}
+[[File:Igku Sep2 ColonyPCR (N1-2).jpg]]<br>
 </div>
@@ Line 31: / Line 50: @@
 ! ||pSB1C3-(1)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
 |-
-|2 cuts(E+S)||7&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+|2 cuts(EcoRI+SpeI)||7&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
 |-
-|NC(E+S)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
+|NC(EcoRI+SpeI)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
 |-
-|2 cut(X+P)s||7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+|2 cuts(XbaI+PstI)||7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
 |-
-|NC(X+P)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
+|NC(XbaI+PstI)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
 |}
 {| class="wikitable"
@@ Line 56: / Line 75: @@
 ! ||8/21 pT181 attenuator-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
 |-
-|2 cuts(E+S)||8&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
+|2 cuts(EcoRI+SpeI)||8&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
 |-
-|NC(E+S)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+|NC(EcoRI+SpeI)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
 |-
-|2 cut(X+P)s||8&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
+|2 cuts(XbaI+PstI)||8&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
 |-
-|NC(X+P)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+|NC(XbaI+PstI)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
 |}
 [[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
@@ Line 109: / Line 128: @@
 |10||Fuaion6 antisense-1
 |}
+</div>
+===Electrophoresis===
+<div class="experiment">
+<span class="author">Nakamoto & Tatsui</span>
+{| class="wikitable"
+!Lane||Sample||Enzyme1||Enzyme2
+|-
+|1||100bp ladder||--||--
+|-
+|2||pSB1C3 -(1)||EcoRI||SpeI
+|-
+|3||pSB1C3 -(1)||--||--
+|-
+|5||pSB1C3 -(1)||XbaI||PstI
+|-
+|6||pSB1C3 -(1)||--||--
+|-
+|7||pT181 attenuator (2)||EcoRI||SpeI
+|-
+|8||pT181 attenuator (2)||--||--
+|-
+|9||pT181 attenuator (2)||XbaI||SpeI
+|-
+|10||pT181 attenuator (2)||--||--
+|-
+|11||tetR-aptamer 12_1R||EcoRI||SpeI
+|-
+|12||tetR-aptamer 12_1R||--||--
+|-
+|13||tRNA Spinach (1)||EcoRI||SpeI
+|-
+|14||100bp ladder||--||--
+|-
+|15||tRNA Spinach (1)||--||--
+|}
+[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
 </div>
@@ Line 119: / Line 175: @@
 |1||100bp ladder||-
 |-
-|2||rowspan="2"|pSB1C3(E+S)||rowspan="2"|EcoRI&SpeI
+|2||rowspan="2"|pSB1C3(EcoRI+SpeI)||rowspan="2"|EcoRI&SpeI
 |-
 |3
@@ Line 131: / Line 187: @@
 [[File:Igku Sep1 Gel Extraction(N2-1).jpg]]<br>
 [[File:Igku Sep1 Gel Extraction(N2-2).jpg]]<br>
-<span class="author">No name</span>
 {| class="wikitable"
 !Lane||DNA||Enzyme
@@ Line 137: / Line 192: @@
 |1||100bp ladder||-
 |-
-|2||rowspan="2"|pT181 attenuator(2)(E+S)||rowspan="2"|EcoRI&SpeI
+|2||rowspan="2"|pT181 attenuator(2)(EcoRI+SpeI)||rowspan="2"|EcoRI&SpeI
 |-
 |3
@@ Line 143: / Line 198: @@
 |4||--||--
 |-
-|5||rowspan="2"|pT181 attenuator(2)(X+P)||rowspan="2"|EcoRI&SpeI
+|5||rowspan="2"|pT181 attenuator(2)(XbaI+PstI)||rowspan="2"|EcoRI&SpeI
 |-
 |6
@@ Line 149: / Line 204: @@
 |7||--||--
 |-
-|8||rowspan="2"|pT181 attenuator(2)(X+P)||rowspan="2"|EcoRI&SpeI
+|8||rowspan="2"|pT181 attenuator(2)(XbaI+PstI)||rowspan="2"|EcoRI&SpeI
 |-
 |9
@@ Line 197: / Line 252: @@
 |5min||30s||30s||40s||30cycles
 |}
-[[File:Igku Sep2 ColonyPCR (N4).jpg]]
 {| class="wikitable"
 !|Sample||base pair
@@ Line 223: / Line 277: @@
 |}
 [[File:Igku Sep3 ColonyPCR (N1).jpg]]
+</div>
+===Electrophoresis===
+<div class="experiment">
+<span class="author">No name</span>
+{| class="wikitable"
+!Lane||Sample
+|-
+|1||100bp ladder
+|-
+|2||Spinach(pSB1C3)
+|-
+|3||Spinach-DT
+|-
+|4||RBS-lysis1-DT
+|-
+|5||100bp ladder
+|}
+[[File:Igku Sep2 ColonyPCR (N4).jpg]]<br>
 </div>

Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

Latest revision as of 18:50, 25 September 2013

Contents

Sep 2

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

Liquid Culture

Master Plate

Electrophoresis

Gel Extraction

Colony PCR

Electrophoresis

Master Plate

Liquid Culture

Transformation