Template:Kyoto/Notebook/Sep 25

From 2013.igem.org

(Difference between revisions)

Revision as of 06:53, 27 September 2013

Sep 25

Electrophoresis

nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-antisense (1C3-66E-fwd A2-662 rev)	--	--
3	DT (1C3-661-fwd 1C3 662 rev)	--	--
4	spinach-DT(1C3-661-fwd 1C3 662 rev)	--	--
5	Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)	--	--
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
2	100bp ladder	--	--
3	Ptet (1C3 66E fwd-1C3 660 rev)	--	--
4	Pcon-attenuator(1C3-660 fwd A2-661 rev)	--	--
5	DT(1C3 662 fwd-1C3 661 rev)	--	--
6	Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)	--	--
7	100bp ladder	--	--

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)	859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)	1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)	601

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
2 min	30 s	30 s	1min5s	30 cycles

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-PT181 attenuator-aptamer12-1R-DT-5	--	--
2	Pcon-PT181 attenuator-aptamer12-1R-DT-6	--	--
3	Pcon-PT181 attenuator-aptamer12-1R-DT-7	--	--
4	Pcon-PT181 attenuator-aptamer12-1R-DT-8	--	--
5	Pcon-PT181 attenuator-aptamer12-1R-DT-9	--	--
6	Pcon-PT181 attenuator-aptamer12-1R-DT-10	--	--
7	Pcon-PT181 attenuator-aptamer12-1R-DT-11	--	--
8	Pcon-PT181 attenuator-aptamer12-1R-DT-12	--	--
9	1kbp ladder	--	--
10	Pcon-PT181 attenuator-RBS-GFP-DT-5	--	--
11	Pcon-PT181 attenuator-RBS-GFP-DT-6	--	--
12	Pcon-PT181 attenuator-RBS-GFP-DT-7	--	--
13	Pcon-PT181 attenuator-RBS-GFP-DT-8	--	--
14	Pcon-PT181 attenuator-pSB1C3-5	--	--
15	Pcon-PT181 attenuator-pSB1C3-6	--	--
16	Pcon-PT181 attenuator-pSB1C3-7	--	--
17	Pcon-PT181 attenuator-pSB1C3-8	--	--

Transformation

kojima and nakamoto

Name	Sample	Competent Cells	Total	Plate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--

Name	Sample	Competent Cells	Total	Plate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)	2µL	20µL	22µL	--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))	2µL	20µL	22µL	--

Ligation

nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/25 aptamer12_1R-DT(EcoRI&XbaI)	4.5 µL	Pcon-pT181 attenuator(EcoRI&SpeI)	7.7 µL	6.1 µL
experiment	9/17 pSB1C3(EcoRI&SpeI)	2.0 µL	9/3 pT181 attenuator(EcoRI&SpeI)	14 µL	8 µL
experiment	9/25 Pcon-pT181 attenuator(EcoRI&XbaI)	4.5 µL	9/25 RBS-GFP-DT(EcoRI&SpeI)	19.3 µL	11.9 µL
experiment	9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)	4.2 µL	Pcon-RBS-tetR-DT(EcoRI&SpeI)	20 µL	12.1 µL

Restriction Enzyme Digestion

nakamoto>

	9/24 Pcon-attenuator-aptamer-DT(µL)	EcoRI(µL)	SpeI(µL)	Buffer(µL)	MilliQ(µL)	total(µL)
2 cuts	8.6	1	1	3	16.4	30
NC	0.4	0	0	1	8.6	10

	9/11 DT(µL)	EcoRI(µL)	XbaI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	10.4	1	1	3	3	11.6	30
NC	0.5	0	0	1	1	7.5	10

Liquid Culture

Kojima

Sample	medium
DT	--
pT181attenuator	--
Pcon-RBS-tetR-DT	--

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcon-pT181antisense	--
3
4
5	--	--
6	DT	--
7
8
9	--	--
10	Spinach-DT	--
11
12

Lane	DNA	Enzyme
1	100bp ladder	--
3	Pcon-antisense-spinach-DT(E-1A)	--
4	Pcon-antisense-spinach-DT(E-1A)	--
5	Pcon-antisense-spinach-DT(E-1A)	--

Lane	DNA	Enzyme
7	100bp ladder	--
9	Ptet(E-0)	--
10	Ptet(E-0)	--
11	Ptet(E-0)	--

Lane	DNA	Enzyme
13	100bp ladder	--
15	Pcon-attenuator(0-1A)	--
16	Pcon-attenuator(0-1A)	--
17	Pcon-attenuator(0-1A)	--

||--

Lane	DNA	Enzyme
19	100bp ladder	--
21	Pcon-attenuator-aptamer-DT(E-1A)	--
22	Pcon-attenuator-aptamer-DT(E-1A)	--
23	Pcon-attenuator-aptamer-DT(E-1A)	--

Name	concentration[µg/mL]	260/280	260/230
DT(2-1)	24.9	1.73	0.37
Ptet(E-0)	12.8	1.59	0.90
Pcon-antisense(E-2A)	16.5	1.72	1.07
Pcon-attenuator-aptamer-DT(E-1A)	22.1	1.75	0.94
Pcon-antisense-spinach-DT(E-1A)	31.0	1.78	1.15
spinach-DT(1-2)	23.4	1.85	1.11
Pcon-attenuator(0-1A)	22.5	1.76	0.46
DT(1-2)	25.1	1.78	1.21

Electrophoresis

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pcon-pT181 attenuator-aptamer 12_1R-DT	EcoRI	SpeI
3	Pcon-pT181 attenuator-aptamer 12_1R-DT	--	--
4	DT	EcoRI	XbaI
5	DT	--	--

Gel Extraction

nakamoto

Lane	DNA	Enzyme
1	DT	EcoRI+XbaI
2	DT	EcoRI+XbaI
3	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
4	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
5	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)	11.8	1.80	0.46
DT(EcoRI+XbaI)	42.6	1.84	0.98

Liquid Culture

No name

Sample	medium
Pcon-pT181anntisense-spinach-DT(4k5)	-

Ligation

Nakamoto and Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL

PCR

hirano

800pg/μL DT	KOD plus	10x buffer	dNTP	MgSO4	1C3_GG2_fwd	iC3_GG1_rev	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	24s	30cycles

Transformation

Tatsui

Name	Sample	Competent Cells	Total	Plate
aptamer12-1R-DT	µL	µL	µL	CP
pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)	µL	µL	µL	CP
Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)	µL	µL	µL	Amp
Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)	µL	µL	µL	Amp
pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)	µL	µL	µL	CP

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	DT(2-1)	--	--

Column Refining

Name	concentration[µg/mL]	260/280	260/230
8/25 DT(2-1)	--	--	--

Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.

	9/25 DT(2-1)	10*cut smart Buffer	BsaI-HF	MilliQ	total
	3.0 µL	2.0 µL	0.3 µL	14.7 µL	20.0 µL

Incubate 37°C 12hour </div>

Liquid Culture

hirano

Sample	medium
Pcon-GFP-DT	LB(Amp)
RBS-GFP-DT	LB(CP)
Pcon-spinach-DT	LB(Amp)
Pcon-tetRaptamer-DT	LB(Amp)
Pcon-attenuator-DT	LB(Amp)
spinach-DT	LB(CP)
Pcon-antisense-spinach-DT	LB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min

OD600-1.445

Sample	Use plate	The number of plate
Pcon-GFP-DT	M9(+Amp)	3
Pcon-GFP-DT	LB(+Amp)	3

</div> incubate 37°C

Colony PCR

No name

Sample	base pair
9/24 GGA-1(1~8)	3177
9/24 GGA-2(1~8)	3189
9/24 GGA-3(1~8)	2989
9/24 GGA-4(1~8)	2939
9/24 GGA-7(1)	2489
GGA-16(1)	2717
RBS-GFP-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min15s	30cycles

RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS-GFP-DT	2783	1.95	1.48

Add 91.2µL, Store at freezer
Total 250µL</div>

cDNA　Synthesis

Nakamoto

volume	7 µL
7*cDNA Wipcout Buffer	1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)	3 µL
RNase-frec water	4 µL

Incubate 42°C 2min, then on ice immediately
Add

5*Quantiscript Reverse Transcriptase	0.5 µL
5*Quantiscript RT Buffer	2 µL
RT Primer Mix	0.5µL

Incubate 42°C 15min, then incubate 95°C 3min

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-RBS-GFP-DT(RNA)	--	--

Ligation

tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-pT181-attenuator(SpeI&PstI)	3.5µL	9/25 RBS-GFP-DT(XbaI&PstI)	13.5µL	8.5 µL

@@ Line 172: / Line 172: @@
 <span class="author">nakamoto>
 {| class="wikitable"
-! ||9/24 Pcon-attenuator-aptamer-DT(&micro;L)||E(&micro;L)||S(&micro;L)||Buffer(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
+! ||9/24 Pcon-attenuator-aptamer-DT(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||Buffer(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 |-
 |2 cuts||8.6||1||1||3||16.4||30
@@ Line 179: / Line 179: @@
 |}
 {| class="wikitable"
-! ||9/11 DT(&micro;L)||E(&micro;L)||X(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
+! ||9/11 DT(&micro;L)||EcoRI(&micro;L)||XbaI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 |-
 |2 cuts||10.4||1||1||3||3||11.6||30

Template:Kyoto/Notebook/Sep 25

From 2013.igem.org

Revision as of 06:53, 27 September 2013

Contents

Sep 25

Electrophoresis

Colony PCR

Electrophoresis

Transformation

Ligation

Restriction Enzyme Digestion

Liquid Culture

Gel Extraction

Electrophoresis

Gel Extraction

Liquid Culture

Ligation

PCR

Transformation

Electrophoresis

Column Refining

Restriction Enzyme Digestion

Liquid Culture

Plating

Colony PCR

RNA Extraction

cDNA Synthesis

Electrophoresis

Ligation

cDNA　Synthesis