Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

(Difference between revisions)

Revision as of 16:28, 27 September 2013

Sep 14

Restriction Enzyme Digestion

Hirano

	9/14 pSB4K5S	EcoRI	XbaI	SpeI	PstI	BufferB	BufferD	BSA	MilliQ	total
2cuts	10µL	1µL	0µL	1µL	0µL	3µL	0µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	1µL	7.5µL	10µL

	9/13 Plac	XbaI	PstI	BufferH	BSA	MilliQ	total
1 cut	9µL	0µL	1µL	3µL	3µL	14µL	30µL
NC	1µL	0µL	0µL	1µL	1µL	7µL	10µL

	9/14 PKaiBC	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	11µL	1µL	0µL	1µL	0µL	3µL	3µL	11µL	30µL
NC	4µL	0µL	0µL	0µL	0µL	1µL	1µL	4µL	10µL

	9/14 RpaB	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	13µL	0µL	1µL	0µL	1µL	3µL	3µL	9µL	30µL
NC	2µL	0µL	0µL	0µL	0µL	1µL	1µL	5µL	10µL

	8/17 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
1cut	9µL	0µL	1µL	0µL	0µL	3µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

incubated 37°C 1hour

Liquid Culture

Hirano

Sample	medium
9/11 Plac(BBa-R0011)-(3)	Plusgrow medium(+Amp)

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	pSB4K5(1)	EcoRI	SpeI
3	pSB4K5(1)	--	--
4	Plac(1A2)	PstI	--
5	Plac(1A2)	--	--
6	PKaiBC	EcoRI	SpeI
7	PKaiBC	--	--
8	RpaB(1)	XbaI	PstI
9	RpaB(1)	--	--
10	RBS-GFP-DT	XbaI	--
11	RBS-GFP-DT	--	--
12	1kb ladder	--	--

File:Igku xxxxxx.xxx

Lane	Sample	Enzyme
1	1kb ladder	--
2	Plac(pSB1A2)	PstI
3	Plac(pSB1A2)	--

File:Igku xxxxxx.xxx
</div>

Sequence PCR

No name

genome DNA	Big Dye	5x buffer	temp(400mg)	primer	milliQ
Pcon-RBS-luxR-DT	0.5	1.75	0.9	0.5	6.85
Pcon-RBS-luxR-DT	0.5	1.75	0.4	0.5	7.35
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Plux	0.5	1.75	2.4	0.5	5.35
Plux-RBS-GFP-DT	0.5	1.75	1.9	0.5	5.85
Plux-RBS-GFP-DT	0.5	0.5	1.9	0.5	5.85
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
RBS-lysis3-DT	0.5	0.5	1.0	0.5	6.75
Pcon-antisense-Spinach-DT	0.5	0.5	1.4	0.5	6.35
Pcon-RBS lacZα-DT	0.5	0.5	1.4	0.5	6.35
Ptet-RBS lacZα-DT	0.5	0.5	0.59	0.5	1.85

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	60°C	--
2min	10sec	5sec	4min	30cycle

Restriction Enzyme Digestion

No name

	9/14 Plac(1A2)	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	23µL	0µL	0µL	0.3µL	0µL	3µL	3µL	0µL	30µL

	9/14 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferH	BSA	MilliQ	total
2 cuts	23µL	1µL	0µL	0µL	0µL	3µL	3µL	0µL	30µL

Ligation

Nakamoto and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181attenuator(SpeI&PstI)	1.7	9/13 RBS-lacZα-DT (XbaI & PstI)	12.5	5
experiment	9/13 pSB2C3 (XbaI & PstI)	1.9	9/14 RpaB (XbaI & PstI)	16.3	10.5
experiment	9/13 pSB2C3 (XbaI & PstI)	4.7	9/14 PkaiBC (EcoRI & SpeI)	6.7	5.7
experiment	9/14 RBS-GFP-DT (PstI&XbaI)	0.7	9/14 PkaiBC (EcoRI & SpeI)	2.6	1.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis1-DT (XbaI & PstI)	4.9	3.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis2-DT (XbaI & PstI)	7.0	4.7
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis3-DT (XbaI & PstI)	9.2	5.8

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Sample	base pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)	--
9/13 Plux＋RBS-lysis2-DT (1)~(3)	-
9/13 Plux＋RBS-lysis3-DT (1)~(2)	-

Sample	base pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)	--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)	-
9/13 Plac＋PT181attenuator (1)	-

Sample	base pair
Dλ-lux(1) (1)~(2)	--
NC	--

Liquid Culture

No name

Sample	medium
9/13 Pλ-luxI(1)-1	Plusgrow medium(+Amp)

Transformation

Nakamoto

Name	Sample	Competent Cells	Total	Plate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)	2µL	20µL	22µL	Amp
9/13 SB2C3＋9/14 PzαB	2µL	20µL	22µL	CP
9/13 pSB2C3＋9/14 PkaiBC	2µL	20µL	22µL	CP
9/13 RBS-GFP-DT＋9/14 PkaiBC	2µL	20µL	22µL	CP
Plac＋RBS-lysis1-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp

PCR

Tatsui

genome DNA	temp	Big Dye	5x buffer	primer	MilliQ	total
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85	10.5
8/17 RBS lacZα-DT	2.1	0.5	1.75	0.5	3.85	10.5
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85	10.5

PreDenature	Denature	Annealing	Extension	cycle
96°C	50°C	60°C	4°C	--
2min	13s	5s	4min	30cycle

@@ Line 51: / Line 51: @@
 </div>
-===PCR===
+===Electrophoresis===
+<div class="experiment">
+<span class="author">Hirano</span>
+{| class="wikitable"
+!Lane||Sample||Enzyme1||Enzyme2
+|-
+|1||1kb ladder||--||--
+|-
+|2||pSB4K5(1)||EcoRI||SpeI
+|-
+|3|| pSB4K5(1)||--||--
+|-
+|4||Plac(1A2)||PstI||--
+|-
+|5||Plac(1A2)||--||--
+|-
+|6||PKaiBC||EcoRI||SpeI
+|-
+|7||PKaiBC||--||--
+|-
+|8||RpaB(1)||XbaI||PstI
+|-
+|9||RpaB(1)||--||--
+|-
+|10||RBS-GFP-DT||XbaI||--
+|-
+|11||RBS-GFP-DT||--||--
+|-
+|12||1kb ladder||--||--
+|}
+[[File:igku_xxxxxx.xxx]]<br>
+</div>
+{| class="wikitable"
+!Lane||Sample||Enzyme
+|-
+|1||1kb ladder||--
+|-
+|2||Plac(pSB1A2)||PstI
+|-
+|3||Plac(pSB1A2)||--
+|}
+[[File:igku_xxxxxx.xxx]]<br>
+</div>
+===Sequence PCR===
 <div class="experiment">
 <span class="author">No name</span>
 {| class="wikitable"
-!genome DNA||MilliQ||Big Dye||5x buffer||temp(400mg)||primer||total
+!genome DNA||Big Dye||5x buffer||temp(400mg)||primer||milliQ
 |-
-|Pcon-RBS-luxR-DT||0.5||1.75||1.75||0.4||0.5||5.1||10.5
+|Pcon-RBS-luxR-DT||0.5||1.75||0.9||0.5||6.85
 |-
-|Pcon-RBS-luxR-DT||0.5||1.75||1.75||0.4||0.5||5.1||10.5
+|Pcon-RBS-luxR-DT||0.5||1.75||0.4||0.5||7.35
 |-
-|Pcon-RBS-tetR-DT||0.5||1.75||1.75||3||0.5||3||10.5
+|Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25
 |-
-|Pcon-RBS-tetR-DT||0.5||1.75||1.75||3||0.5||3||10.5
+|Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25
 |-
-|Pcon-RBS-luxR-DT||0.5||1.75||1.75||2.4||0.5||3.6||10.5
+|Plux||0.5||1.75||2.4||0.5||5.35
 |-
-|Plux-RBS-GFP-DT||0.5||1.75||1.75||1.9||0.5||4.1||10.5
+|Plux-RBS-GFP-DT||0.5||1.75||1.9||0.5||5.85
 |-
-|Plux-RBS-GFP-DT||0.5||1.75||0.5||1.9||0.5||4.1||10.5
+|Plux-RBS-GFP-DT||0.5||0.5||1.9||0.5||5.85
 |-
-|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.2||0.5||4.8||10.5
+|Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55
 |-
-|RBS-lysis3-DT||0.5||1.75||0.5||1.0||0.5||5.0||10.5
+|Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55
 |-
-|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.4||0.5||4.6||10.5
+|RBS-lysis3-DT||0.5||0.5||1.0||0.5||6.75
 |-
-|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.4||0.5||4.6||10.5
+|Pcon-antisense-Spinach-DT||0.5||0.5||1.4||0.5||6.35
 |-
-|RBS-lysis3-DT||0.5||1.75||1.75||0.4||0.5||5.1||10.5
+|Pcon-RBS lacZ&alpha;-DT||0.5||0.5||1.4||0.5||6.35
 |-
-|Ptet-RBS-spinach-DT||0.5||1.75||0.5||0.59||0.5||0.1||10.5
+|Ptet-RBS lacZ&alpha;-DT||0.5||0.5||0.59||0.5||1.85
 |}
 {| class="wikitable"
@@ Line 88: / Line 134: @@
 |96&deg;C||96&deg;C||50&deg;C||60&deg;C||--
 |-
-|2min||10s||5s||4min||30cycle
+|2min||10sec||5sec||4min||30cycle
 |}
 </div>

Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

Revision as of 16:28, 27 September 2013

Contents

Sep 14

Restriction Enzyme Digestion

Liquid Culture

Electrophoresis

Sequence PCR

Restriction Enzyme Digestion

Ligation

Colony PCR

Liquid Culture

Transformation

PCR