Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

(Difference between revisions)

Revision as of 10:53, 25 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)

PCR

No name

genome DNA	MilliQ	Big Dye	5x buffer	temp(400mg)	primer	total
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	1.75	0.4	0.5	5.1	10.5
Pcon-RBS-tetR-DT	0.5	1.75	1.75	3	0.5	3	10.5
Pcon-RBS-tetR-DT	0.5	1.75	1.75	3	0.5	3	10.5
Pcon-RBS-luxR-DT	0.5	1.75	1.75	2.4	0.5	3.6	10.5
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	0.5	1.9	0.5	4.1	10.5
Pcon-RBS-GFP-DT	0.5	1.75	0.5	1.2	0.5	4.8	10.5
RBS-lysis3-DT	0.5	1.75	0.5	1.0	0.5	5.0	10.5
Pcon-RBS-GFP-DT	0.5	1.75	0.5	1.4	0.5	4.6	10.5
Pcon-RBS-GFP-DT	0.5	1.75	0.5	1.4	0.5	4.6	10.5
RBS-lysis3-DT	0.5	1.75	1.75	0.4	0.5	5.1	10.5
Ptet-RBS-spinach-DT	0.5	1.75	0.5	0.59	0.5	0.1	10.5

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

Restriction Enzyme Digestion

No name

	8/22 psB1C3-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts	8µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	17.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	7.9µL	10µL
2 cuts	8µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	17.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/21 tRMA-spinach(1)	XbaI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	4µL	0.5µL	0.5µL	3µL	0.3µL	21.7µL	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	3µL	0.5µL	0.5µL	3µL	0.3µL	22.7µL	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	4µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.4µL	10µL
2 cuts	4µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/21 PT181 antisense-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts	10µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	15.7µL	30µL
NC	1µL	0µL	0&;L	0µL	0µL	1µL	0µL	0.1µL	7.9µL	10µL
2 cuts	10µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	15.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

incubated 37°C 1hour

Ligation

Nakamoto and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	8/30 pSB1C3 (EcoRI & SpeI)	6.6	8/30 tRNA-Spinach (EcoRI & SpeI)	12.5	3.5
experiment	8/30 pSB1C3 (EcoRI & SpeI)	6.6	8/30 pT181 antisense (EcoRI & SpeI)	12.5	3.5
experiment	8/30 pSB1C3 (XbaI & Pst)	12.5	8/30 pT181 attenuator (XbaI & PstI)	25	3.5
experiment	8/30 pSB1C3 (XbaI & Pst)	12.5	8/30 pT181 antisense (XbaI & PstI)	25	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 pT181 attenuator(2) (EcoRI & SpeI)	12.5	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 pT181 antisense (EcoRI & SpeI)	12.5	3.5
experiment	8/27 DT (EcoRI & XbaI)	3.0	8/30 tRNA-Spinach (EcoRI & SpeI)	12.5	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 RBS-lysis2 (EcoRI & SpeI)	12.5	3.5
experiment	8/22 J23100 (SpeI & PstI)	--	RBS-tetR-DT (XbaI & PstI)	--	3.5
experiment	8/22 Plac (SpeI & PstI)	8.0	8/18 RBS-lacZα-DT (XbaI & PstI)	10	3.5

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Sample	base pair
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)	2143
8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)	1227
8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)	2256
8/29 RBS-lysis3-DT-(1)	1210

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycle

Liquid Culture

No name

Sample	medium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)	Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)	Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)	Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)	Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)	Plusgrow medium(+CP)
RBS-lysis3-DT-(1)	Plusgrow medium(+CP)

Transformation

No Name

Name	Sample	Competent Cells	Total	Plate
8/27 pT181 attenuator-DT	2µL	20µL	22µL	CP
8/27 RBS-lysis3-DT	2µL	20µL	22µL	CP
8/28 RBS-lysis3-DT	2µL	20µL	22µL	CP
8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT	2µL	20µL	22µL	Amp
8/28 RBS-lysis1-DT	2µL	20µL	22µL	CP
8/28 Plux-RBS-GFP-DT	2µL	20µL	22µL	CP
8/28 Spinach-DT	2µL	20µL	22µL	CP

File:Igku Aug29 Transformation()after

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
	0.5	2.5	2.5	1.5	0.75	0.75		25

PreDenature	Denature	Annealing	Extension	cycle
°C	°C	°C	°C	--
（◎Д◎）

@@ Line 52: / Line 52: @@
 ===PCR===
-<!-- ここから -->
 <div class="experiment">
 <span class="author">No name</span>
 {| class="wikitable"
-!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+!genome DNA||MilliQ||Big Dye||5x buffer||temp(400mg)||primer||total
 |-
-| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+|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||1.75||0.4||0.5||5.1||10.5
+|-
+|Pcon-RBS-tetR-DT||0.5||1.75||1.75||3||0.5||3||10.5
+|-
+|Pcon-RBS-tetR-DT||0.5||1.75||1.75||3||0.5||3||10.5
+|-
+|Pcon-RBS-luxR-DT||0.5||1.75||1.75||2.4||0.5||3.6||10.5
+|-
+|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||0.5||1.9||0.5||4.1||10.5
+|-
+|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.2||0.5||4.8||10.5
+|-
+|RBS-lysis3-DT||0.5||1.75||0.5||1.0||0.5||5.0||10.5
+|-
+|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.4||0.5||4.6||10.5
+|-
+|Pcon-RBS-GFP-DT||0.5||1.75||0.5||1.4||0.5||4.6||10.5
+|-
+|RBS-lysis3-DT||0.5||1.75||1.75||0.4||0.5||5.1||10.5
+|-
+|Ptet-RBS-spinach-DT||0.5||1.75||0.5||0.59||0.5||0.1||10.5
 |}
 {| class="wikitable"
 !PreDenature||Denature||Annealing||Extension||cycle
 |-
-|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+|96&deg;C||96&deg;C||50&deg;C||60&deg;C||--
 |-
-|（◎Д◎）　||　||　||　||
+|2min||10s||5s||4min||30cycle
 |}
 </div>
-<!-- ここまでをコピーしてね -->
 ===Restriction Enzyme Digestion===

Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

Revision as of 10:53, 25 September 2013

Contents

Sep 14

Restriction Enzyme Digestion

Liquid Culture

PCR

Restriction Enzyme Digestion

Ligation

Colony PCR

Liquid Culture

Transformation

PCR