Template:Kyoto/Notebook/Aug 20

From 2013.igem.org

Aug 20

Miniprep

Kojima

DNA	concentration[µg/µL]	260/280	260/230
8/18 Ptet -1	220	1.69	1.87
8/18 Ptet -2	210	1.69	1.71
8/18 RBS-tetR-DT -1	236	1.63	1.69
8/18 RBS-tetR-DT -2	182	1.65	1.98
8/18 J23100-RBS-GFP-DT -1	334	1.71	2.12
8/18 J23100-RBS-luxR-DT -1	440	1.67	1.60
8/18 J23100-RBS-luxR-DT -2	344	1.68	1.83
8/18 J23100-RBS-lacZα-DT -1	280	1.70	1.81
8/18 RBS-lysis3 -1	282	1.67	1.76
8/18 RBS-lysis3 -2	212	1.30	1.35

LB Medium Plate

Okazaki

volume	200mL
Bacto Trypton	2g
Bacto yeast extract	1g
Nacl	1g
Agar Powder	2g
Amp	40µl

1. Measuring reagents and put they in the Erlenmeyer flask.
2. diluting in measuring cylinder to 200ml total.
3. autoclaving.
4. adding ampicirine 40ml.

Ristriction Enzyme Digestion

Kojima and Ashida

	8/20 Ptet -1(220µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	4.5	1	1	3	3	17.5	30
1 cut	0.5	0.2	0	1	1	7.3	10
1 cut	0.5	0	0.2	1	1	7.3	10
NC	0.5	0	0	1	1	7.5	10

	8/20 RBS-tetR-DT -2(182µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	5.5	1	1	3	19.5	30
1 cut	0.6	0.2	0	1	8.2	10
1 cut	0.6	0	0.2	1	8.2	10
NC	0.6	0	0	1	8.4	10

	8/20 J23100-RBS-GFP-DT(334µg/ml)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	3.0	1	1	3	22	30
1 cut	0.3	0.2	0	1	8.5	10
1 cut	0.3	0	0.2	1	8.5	10
NC	0.3	0	0	1	8.7	10

	8/20 J23100-RBS-luxR-DT -2(344µg/mL)	EcoRI	XbaI	BSA	10x buffer M	MilliQ	total
2 cuts	2.9	1	1	3	3	19.1	30
1 cut	0.3	0.2	0	1	1	7.5	10
1 cut	0.3	0	0.2	1	1	7.5	10
NC	0.3	0	0	1	1	7.7	10

	8/20 RBS-lysis3 -1(282µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	3.5	1	1	3	21.5	30
1 cut	0.4	0.2	0	1	8.4	10
1 cut	0.4	0	0.2	1	8.4	10
NC	0.4	0	0	1	8.6	10

	8/17 DT -1(188µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	5.3	1	1	3	3	16.7	30
1 cut	0.3	0.2	0	1	1	7.5	10
1 cut	0.3	0	0.2	1	1	7.5	10
NC	0.3	0	0	1	1	7.7	10

Transformation

Nakamoto

Name	Sample	Competent Cells(XL10-gold)	Total	Plate
tRNA-Spinach-tRNA	1µL	10µL	11µL	Amp
tetR-aptamer 12_PC(076)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1R(113)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1M(105)	1µL	10µL	11µL	Amp
pT181 attenuator	1µL	10µL	11µL	Amp
Fusin1 attenuator	1µL	10µL	11µL	Amp
Fusion3m2 attenuator	1µL	10µL	11µL	Amp
pT181 antisense	1µL	10µL	11µL	Amp
Fusion1 antisense	1µL	10µL	11µL	Amp
Fusion6 antisense	1µL	10µL	11µL	Amp

incubate 37°C overnight 20:34~

Electrophoresis