Team:Kyoto/Notebook1

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Revision as of 14:02, 23 October 2010

LastModified: 2010-10-23

Notebook

Construction for Lysisbox

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Incubation	Colony
J23100	1-18-C	1 µL	20	21	LB (Amp+)	At 37℃, 7/20 20:50 - 7/21 17:00	○
J23105	1-18-M	1	20	21			○
J23116	1-20-M	1	20	21			○
R0011	1-6-G	1	20	21			○
E0840	1-12-O	1	20	21			○
J06702	2-8-E	1	20	21			○
pSB4K5	1-5-G	1	20	21			×
B0015	1-23-L	1	20	21	LB (Kan+)		×

A vector of pSB4K5 is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture B0015 despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of pSB4K5 and B0015.

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Incubation	Colony
pSB4K5	1-5-G	1 µL	20	21	LB (Kan+)	At 37℃, 7/21 20:50 - 7/22 16:30	○
B0015	1-23-L	1	20	21	LB (Kan+)	At 37℃, 7/21 20:50 - 7/22 16:30	○

PCR for SRRz and S

No.	Water	MgSO4	dNTPs	10xBuffer	Template DNA	Primer Fwd.	Primer Rev. (SRRz)	Primer Rev. (S)	KOD Plus ver.2	Total
1	28 µL	3	5	5	5	1.5	1.5	-	1	50
2	28	3	5	5	5	1.5	1.5	-	1	50
3	28	3	5	5	5	1.5	-	1.5	1	50
4	28	3	5	5	5	1.5	-	1.5	1	50
5	28	3	5	5	5	1.5	1.5	-	1	50
6	28	3	5	5	5	1.5	1.5	-	1	50
7	28	3	5	5	5	1.5	-	1.5	1	50
8	28	3	5	5	5	1.5	-	1.5	1	50

94℃	2min
98℃	10s	30 cycles
55℃	30s
68℃	4min
4℃	forever

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

No.	Name	Length(bp)	Result
1	SRRz	1386	○
2	SRRz	1386	○
3	S	442	○
4	S	442	○
5	SRRz	1386	○
6	SRRz	1386	○
7	S	442	○
8	S	442	○

Marker: 100bp, 1kb, 1kb, 100bp.

Miniprep

Name	Concentration
J23100	18.5 (ng/µL)
J23105	12.5
J23116	14.6
R0011	8.6
E0840	12.1
J06702	14.7

The concentration of all samples was very week. Probably our shaking incubation was week.

Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of pSB4K5 and B0015

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

Name	Concentration
pSB4K5	79.2 (ng/µL)
B0015	-

We lost B0015 by our mistake. The concentration of pSB4K5 is high, so this condition of shaking incubation is moderate.

PCR Purification

No.	Name	Concentration	New Name
1	SRRz	18.6 ng/µL	-
3	S	77.6	S_Sam7(1)
5	SRRz	33.6	-
7	S	65.4	S_Sam7(2)

The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

PCR for SRRz

No.	Water	MgSO4	dNTPs	10xBuffer	Template DNA	Primer Fwd. (SRRz)	Primer Rev. (SRRz)	KOD plus ver.2	Total
1	28 µL	3	5	5	5	1.5	1.5	1	50
2	28	3	5	5	5	1.5	1.5	1	50
3	26.5	4.5	5	5	5	1.5	1.5	1	50
4	26.5	4.5	5	5	5	1.5	1.5	1	50
5	25	6	5	5	5	1.5	1.5	1	50
6	25	6	5	5	5	1.5	1.5	1	50

94℃	2min
98℃	10s	30 cycles
55℃	30s
68℃	4min
4℃	forever

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme

No.	Name	Sample	10xBuffer	BSA	Enzyme		MilliQ	Total	Incubation
1	J06702	5 µL	1	0.1	EcoRI	0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	J06702	5	1	0.1	XbaI	0.1	3.6	10
3	J06702	5	1	0.1	SpeI	0.1	3.6	10
4	J06702	5	1	0.1	PstI	0.1	3.6	10
5	J06702	5	1	0.1	-		3.7	10

Marker: 1kb. Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

Restriction Digestion and Ligation to insert S gene to E0840

Name	Sample	10xBuffer	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
S_Sam7(1)	11 µL	5	EcoRI	0.2	SpeI	0.2	33.6	50	At 37℃ for 2h
S_Sam7(2)	11	5	EcoRI	0.2	SpeI	0.2	33.6	50
E0840	45	5	EcoRI	0.2	XbaI	0.2	0	50

After PCR Purification, evaporated them and diluted 3µL.

Name	Vector		Insert		Ligation High	Total
S_Sam7(1)-E0840	E0840	0.5µL	S_Sam7(1)	0.5	1	2
S_Sam7(2)-E0840	E0840	0.5	S_Sam7(2)	0.5	1	2

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

No.	Name	Length(bp)
1	SRRz	1386
2	SRRz	1386
3	SRRz	1386
4	SRRz	1386
5	SRRz	1386
6	SRRz	1386

Marker: 1kb. At the condition 4 (4.5µL MgSO4) and 6 (6µL MgSO4), SRRz is amplified very much. So we decided to use them.

PCR Purification

No.	Name	Concentration	New Name
4	SRRZ	51.6 ng/µL	SRRz_Sam7(1)
5	SRRZ	59.3
6	SRRZ	59.6	SRRz_Sam7(2)

Transformation

Name	Well	Sample	Competent Cell	Total	Plate	Incubation	Colony
E0240	1-12-M	1 µL	20	21	LB (Amp+)	At 37℃ 7/26 - 7/27	×
I20260	2-17-F	1	20	21	LB (Kan+)		×
J04450	1-5-E	1	20	21	LB (Kan+)		×

Culture of pSB4K5, E0840, and B0015

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Colony PCR of S_Sam7-E0840 (Electrophoresis for 35min)

No.	Name	Length	Result
1	S_Sam7(1)-E0840	1522	○
2	S_Sam7(1)-E0840	1522	×
3	S_Sam7(1)-E0840	1522	○
4	S_Sam7(1)-E0840	1522	×
5	S_Sam7(1)-E0840	1522	○
6	S_Sam7(1)-E0840	1522	◎ (Use as S_Sam7(1)-E0840)
7	S_Sam7(2)-E0840	1522	×
8	S_Sam7(2)-E0840	1522	×
9	S_Sam7(2)-E0840	1522	×
10	S_Sam7(2)-E0840	1522	×
11	S_Sam7(2)-E0840	1522	◎ (Use as S_Sam7(2)-E0840)
12	S_Sam7(2)-E0840	1522	○
13	S_Sam7(2)-E0840	1522	○
+	E0840	1116
-	None

Marker: 1kb, 100bp

Miniprep

Name	Concentration
R0011	26.9 ng/µL
B0015	120.0
E0840	120.1

Restriction Digestion

Name	Sample	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
B0015	30 µL	5	0.5	EcoRI	0.4	XbaI	0.3	13.7	50	At 37℃ 16:45 - 18:00
SRRz_Sam7(1)	40	5	0.5	EcoRI	0.4	SpeI	0.4	3.8	50
SRRz_Sam7(2)	40	5	0.5	EcoRI	0.4	SpeI	0.4	3.8	50

Ligation

Transformation

Name	Sample	Competent Cells	Total	Plate	Incubation	Colony
SRRz_Sam7(1)-B0015						○
SRRz_Sam7(2)-B0015						○

Wednesday, July 28 By:

Miniprep

Name	Concentration
S_Sam7(1)-E0840	95.5 ng/µL
S_Sam7(2)-E0840	98.6

Diluted S_Sam7(1)-E0840 and S_Sam7(2)-E0840 20 times with water, and used as template DNA.

Deletion PCR to delete a functional domain of S gene

	Water	MgSO4	dNTPs	10xBuffer	Primer Fwd.	Primer Rev.	Template (1)	Template (2)	KOD Plus ver.2	Total
S_Sam7,ΔTMD1(1)-E0840 (1)	28 µL	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(1)-E0840 (2)	28	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(2)-E0840 (1)	28	3	5	5	1.5	1.5	-	5	1	50
S_Sam7,ΔTMD1(2)-E0840 (2)	28	3	5	5	1.5	1.5	-	5	1	50

94℃	2min
98℃	10s	35 cycles
55℃	30s
68℃	4min
4℃	forever

Restriction Digestion to check the function of DpnI

Name	Sample	fast digestion buffer	DpnI	MilliQ	Total
S_Sam7(1)-E0840	3 µL	1	0.1	5.8	10
S_Sam7(2)-E0840	3	1	0.1	5.8	10

Electrophoresis for 35min

No.	Name	Length
1	Not digested S_Sam7(1)-E0840	3363bp
2	Not digested S_Sam7(2)-E0840	3363
3	Digested S_Sam7(1)-E0840	1021, 933, 402, 341, 258, 105, ...
4	Digested S_Sam7(2)-E0840	1021, 933, 402, 341, 258, 105, ...

Marker: 1kb, 100bp DpnI works correctly.

Thursday, July 29 By:

Restriction Digestion

Name	Sample volume	Fastdigestion Buffer	Enzyme 1		MilliQ	Total	Incubation
S_Sam7,ΔTMD1(1)-E0840 (1)	50 µL	6	DpnI	0.2	3.8	60	07/29 09:40 - 07/29 11:00
S_Sam7,ΔTMD1(2)-E0840 (1)	50	6	DpnI	0.2	3.8	60	07/29 09:40 - 07/29 11:00

Ligation and Phosphorylation

Name	Sample	MilliQ	Ligation High	T4 Kinase	Total	Incubation
S_Sam7,ΔTMD1(1)-E0840 (1)	2 µL	7	5	1	15	07/29 11:30 ~ 07/29 13:00
S_Sam7,ΔTMD1(2)-E0840 (1)	2	7	5	1	15	07/29 11:30 ~ 07/29 13:00

Transformation

Name	Sample Volume	Competent Cell	Total	Plate	Incubation	Colony
S_Sam7,ΔTMD1(1)-E0840 (1)	3 µL	30	33	LB (Amp+)	07/29 ~ 07/30	○
S_Sam7,ΔTMD1(2)-E0840 (1)	3	30	33	LB (Amp+)	07/29 ~ 07/30	○

Monday, August 2 By: Wataru, Ken

Miniprep

Name	Concentration
S_Sam7,ΔTMD1-E0840 (1)	52.7 ng/µL
S_Sam7,ΔTMD1-E0840 (2)	54.4
S_Sam7,ΔTMD1-E0840 (3)	89.5
pSB4K5	50.7
R0011	18.6

PCR of E0240

E0240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.

Name	Water	MgSO4	dNTPs	10xBuffer	Primer VF2	Primer VR	Template E0240	KOD Pllus ver.2	Total
E0240(1)	28 µL	3	5	5	1.5	1.5	5	1	50
E0240(2)	28	3	5	5	1.5	1.5	5	1	50

94℃	2min
98℃	10s	35 cycles
55℃	30s
68℃	4min
4℃	forever

Electrophoresis

PCR Purification

Name	Concentration
E0240(1)	42.6 ng/µL
E0240(2)	55.3

Restriction Digestion for inserting E0240 to pSB4K5 by 3A assembly

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
E0240(1) [XP]	30 µL	5	0.5	XbaI	0.2	PstI	0.2	14.1	50
E0240(2) [XP]	30	5	0.5	XbaI	0.2	PstI	0.2	14.1	50

PCR Purification

Name	Concentration	Volume
E0240(1) [XP]	21.8 ng/µL	40 µL
E0240(2) [XP]	32.4	45

Stored at -20℃.

Error PCR

Name	Water	MgSO4	dNTPs	10xBuffer	Primer VF2	Primer VR	Template (1)	Template (2)	Template (3)	KOD Pllus ver.2	Total
S_Sam7,ΔTMD1-E0840 (1)	32 µL	3	5	5	1.5	1.5	1	-	-	1	50
S_Sam7,ΔTMD1-E0840 (2)	32	3	5	5	1.5	1.5	-	1	-	1	50
S_Sam7,ΔTMD1-E0840 (3)	32	3	5	5	1.5	1.5	-	-	1	1	50

94℃	2min
98℃	10s	20 cycles
68℃	4min	20 cycles
4℃	forever

Restriction Digestion of S_Sam7,ΔTMD1-E0840 by DpnI

Transformation

Name	Sample	Competent Cells	Total	Plate	Incubation	Colony
S_Sam7,ΔTMD1-E0840 (1)	2 µL	20	22	-	-	○
S_Sam7,ΔTMD1-E0840 (2)	2	20	22			}
S_Sam7,ΔTMD1-E0840 (3)	2	20	22			○

Tuesday, August 3 By:

Culture

Picked two colonies from S_Sam7,ΔTMD1-E0840 (1), and S_Sam7,ΔTMD1-E0840 (3), and cultured at 37℃ from 08/03 to 08/04.

Miniprep

Name	Concentration
pSB4K5	60.7 ng/µL
R0011	26.8

Restriction Digestion

Name	Sample	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
R0011 [ES]	50 µL	6	0.6	EcoRI	0.2	SpeI	0.2	3	60
pSB4K5 [EP]	50	6	0.6	EcoRI	0.2	PstI	0.2	3	60
E0240(1) [XP]	50	6	0.6	XbaI	0.2	PstI	0.2	3	60
E0240(2) [XP]	50	6	0.6	XbaI	0.2	PstI	0.2	3	60

PCR Purification

Name	Concentration
pSB4K5 [EP]	39.5 ng/µL
E0240(1) [XP]	21.8
E0240(2) [XP]	32.4

pSB4K5 [EP] is concentrated 10µL and E0240(1) [XP], E0240(2) [XP] are concentrated 1µL.

Ethanol Precipitation

After ethanol precipitation, we diluted pSB4K5 by 2µL MilliQ

Ligation

Name	Vector	Insert 1		Insert 2		Ligation High	Total	Incubation
R0011-E0240(1) [pSB4K5]	pSB4K5 [EP]	1	R0011 [ES]	1	E0240(1) [XP]	1	3	15	17:30 - 20:20
R0011-E0240(2) [pSB4K5]	pSB4K5 [EP]	1	R0011 [ES]	1	E0240(2) [XP]	1	3	15	17:30 - 20:20

PCR of I20260

Name	Water	MgSO4	dNTPs	10xBuffer	Primer VF2	Primer VR	Template I20260	KOD plus ver.2	Total
I20260 (1)	32µL	3	5	5	1.5	1.5	1	1	50
I20260 (2)	32	3	5	5	1.5	1.5	-	1	50

94℃	2min
98℃	10s	30 cycles
55℃	30s
68℃	4min
4℃	forever

PCR Purification

Name	Concentration
I20260	40.6 ng/µL

Restriction Digestion

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
I20260 [EP]	45 µL	6	0.6	EcoRI	0.2	PstI	0.2	8	60

PCR Purification

Name	Concentration	Volume
I20260 [EP]	74.1 ng/µL	30

I20260 [EP] is concentrated at 7µL

Ligation

	Vector	Insert		Ligation High		Total	Incubation
I20260 [pSB4K5]	pSB4K5 [EP]	1	I20260 [EP]	1	2	4	20:00-20:30

Transformation

Name	Sample	Competent Cell	Total	Plate	Incubation	Colony
R0011-E0240(1) [pSB4K5]	1 µL	20	21	LB (Kan+)	08/03-08/04	○
R0011-E0240(2) [pSB4K5]	1	20	21			○
I20260 [pSB4K5]	1	20	21			○

Thursday, August 5 By:

Culture and Master Plates

pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts.

However, white colonies and green colonies are observed in R0011-E0240(1) [pSB4K5] and R0011-E0240(2) [pSB4K5] plate. We cultured both white and green colonies.

In I20260 [pSB4K5], Many of colonies are red, but green colonies are observed. We cultured green colonies.

Name	Color	Incubation
R0011-E0240(1) [pSB4K5] (1)	Green Colony	8/5-8/6
R0011-E0240(1) [pSB4K5] (2)	Green Colony
R0011-E0240(1) [pSB4K5] (3)	White Colony
R0011-E0240(1) [pSB4K5] (4)	White Colony
R0011-E0240(2) [pSB4K5] (1)	Green Colony
R0011-E0240(2) [pSB4K5] (2)	White Colony
R0011-E0240(2) [pSB4K5] (3)	White Colony
R0011-E0240(2) [pSB4K5] (4)	White Colony
I20260 [pSB4K5] (1)	Green Colony
I20260 [pSB4K5] (2)	Green Colony
I20260 [pSB4K5] (3)	Green Colony

Sequence

Name	Concentration
S_ΔTMD1-E0840(1) A	28.9 ng/µL
S_ΔTMD1-E0840(1) B	25.3
S_ΔTMD1-E0840(3) A	26.6
S_ΔTMD1-E0840(3) B	24.0

As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.

Friday, August 6

Miniprep

Name
R0011-E0240(1) [pSB4K5] (1)
R0011-E0240(1) [pSB4K5] (2)
R0011-E0240(1) [pSB4K5] (3)
R0011-E0240(1) [pSB4K5] (4)
R0011-E0240(2) [pSB4K5] (1)
R0011-E0240(2) [pSB4K5] (2)
R0011-E0240(2) [pSB4K5] (3)
R0011-E0240(2) [pSB4K5] (4)
I20260 [pSB4K5] (1)
I20260 [pSB4K5] (2)
I20260 [pSB4K5] (3)

Restriction Digestion

Name	Sample	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
R0011-E0240(1) [pSB4K5] (1) [EP]	50 µL	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(1) [pSB4K5] (2) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(1) [pSB4K5] (3) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(1) [pSB4K5] (4) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(2) [pSB4K5] (1) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(2) [pSB4K5] (2) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(2) [pSB4K5] (3) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240(2) [pSB4K5] (4) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
I20260 [pSB4K5] (1) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
I20260 [pSB4K5] (2) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
I20260 [pSB4K5] (3) [EP]	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60

==Electrophoresis

No.	Name	Length	Results
1	I20260 [pSB4K5] (1) [EP]	960, 4339
2	I20260 [pSB4K5] (2) [EP]	960, 4339
3	I20260 [pSB4K5] (3) [EP]	960, 4339
4	R0011-E0240(1) [pSB4K5] (1) [EP]	980 3378	○
5	R0011-E0240(1) [pSB4K5] (2) [EP]	980 3378	○
6	R0011-E0240(1) [pSB4K5] (3) [EP]	980 3378	}
7	R0011-E0240(1) [pSB4K5] (4) [EP]	980 3378	}
8	R0011-E0240(2) [pSB4K5] (1) [EP]	980 3378	○
9	R0011-E0240(2) [pSB4K5] (2) [EP]	980 3378	}
10	R0011-E0240(2) [pSB4K5] (3) [EP]	980 3378	}
11	R0011-E0240(2) [pSB4K5] (4) [EP]	980 3378	}
12	I20260 [pSB4K5] (1) [EP]	960, 4339	○
13	I20260 [pSB4K5] (2) [EP]	960, 4339	○

White colonies are not inserted R0011 but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of lacI gene.

Error PCR (Retry)

Name	Water	MgSO4	dNTPs	10xBuffer	Primer VF2	Primer VR	Template S_Sam7,ΔTMD1-E0840 failed (50ng/µL)	KOD plus ver.2	Total
S_Sam7,ΔTMD1-E0840 (1)	32	3	5	5	1.5	1.5	1	1	50
S_Sam7,ΔTMD1-E0840 (2)	32	3	5	5	1.5	1.5	1	1	50

94℃	2min
98℃	10s	25 cycles
68℃	4min	25 cycles
Add DpnI 2µL
Incubate	1h
4℃	forever

Transformation

Name	Well	Sample	Competent Cell	Total	Plate	Incubation	Colony
S_Sam7,ΔTMD1-E0840 (1)	-	4 µL	50	54	LB (Kan+)	08/06-08/09	○
S_Sam7,ΔTMD1-E0840 (2)	-	4	50	54			○
I20260	2-17-F	2	50	52			○
	2-I-5	2	50	52	LB (Amp+)		○

Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep

Name	concentration
I20260 [pSB4K5]	116.2 ng/µL
R0011-E0240 [pSB4K5]	146.6

Transfotrmation

Sample	Sample	Competent Cell		Total	Plate	Incuvation	Results
I20260 [pSB4K5]	2 µL	KRX	50	52	LB (Kan+)	08/09 18:00-08/10 12:00	○
R0011-E0240 [pSB4K5]	2	KRX	50	52	LB (Kan+)	08/09 18:00-08/10 12:00	○

Restriction Eigestion and Ethanol Precipitation

To use R0011 for next ligation, we digested it by EcoRI and PstI

Name	Sample	10x Buffer	BSA	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
R0011 [EP]	50	6	0.6	Enzyme 1	EcoRI	0.5	PstI	0.5	2.4	60	At 37℃ 08/09 16:20-18:20

After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation

Sample	Competent cell		Total	Plate	Incuvation	Colony
R0011 [pSB4K5, KRX]	2 µL	KRX	50	52	LB (Kan+)	08/09 20:00-08/10 09:00	○
R0011 [pSB4K5</partrinfo>, C2]	2	C2	50	52	LB (Kan+)	08/09 20:00-08/10 09:00	○

====Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka

Culture

Cultured I20260 [pSB4K5, R0011-E0240 [pSB4K5], R0011 [pSB4K5, KRX], and R0011 [pSB4K5</partrinfo>, C2].

Minprep

Name	Concentration
S_Sam7,ΔTMD1-E0840 (1-1)	9.9 ng/µL
S_Sam7,ΔTMD1-E0840 (1-2)	27.3
S_Sam7,ΔTMD1-E0840 (2-1)	43.2
S_Sam7,ΔTMD1-E0840 (2-2)	34.7

Culture and Master Plate

At 37℃ 08/09 18:00-08/10 9:00

Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya

No.	Medium	Cloud	Incubation
1	Kanamycin	○	At 37℃, 08/10 20:00-08/11 9:00
1	Ampicillin	}
2	Kanamycin	○
2	Ampicillin	○
3	Kanamycin	○
3	Ampicillin	}
4	Kanamycin	○
4	Ampicillin	}
5	Kanamycin	○
5	Ampicillin	}
6	Kanamycin	○
6	Ampicillin	○
7	Kanamycin	○
7	Ampicillin	}

Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.

Miniprep of R0011 [pSB4K5, C2], SRRz 1', 3'

Name	Concentration
R0011 [pSB4K5, C2] (1)	31.2 ng/µL
R0011 [pSB4K5, C2] (3)	29.9

Restriction Digestion and electrophoresis of R0011 [pSB4K5, C2]

Name	EcoRI	PstI
1	0.2	-
2	-	0.2
3	0.2	0.2
N	-	-

No.	Name	Length	Results
1	R0011 [pSB4K5, C2] (1-1)
2	R0011 [pSB4K5, C2] (1-2)
3	R0011 [pSB4K5, C2] (1-3)
4	R0011 [pSB4K5, C2] (1-N)
5	R0011 [pSB4K5, C2] (2-1)
6	R0011 [pSB4K5, C2] (2-2)
7	R0011 [pSB4K5, C2] (2-3)
8	R0011 [pSB4K5, C2] (2-N)

Each enzyme correctly cut samples.

Screening PCR of SRRz

No.	Name	Results
1	None
2	Control B0015
3	Control J06702
4	Control B0015
5-24	SRRz-B0015	}

Marker: Lambda Marker

Discussion: All of the sample were self-ligation of DT. SRRz weren't inserted.

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of B0015

Name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Maker: Lambda, 100bp

Discussion: Each enzyme correctly cut each sample and was active.

====Thursday, August 19 By: Wataru, Tomo, Ken

Miniprep of S_Sam7,ΔTMD1-E0840

Name	Concentration
S_Sam7,ΔTMD1-E0840	29.6 ng/µL

Point mutation PCR of S_Sam7,ΔTMD1-E0840

Name	Template	10xbuffer	dNTPs	MgSO4	Primer Fwd.	Primer Rev.	MilliQ	KOD plus ver.2	Total
S_ΔTMD1-E0840 (1)	1.5	5	5	3	1.5	1.5	31.5	1	50
S_ΔTMD1-E0840 (2)	1.5	5	5	3	1.5	1.5	31.5	1	50
Control	1.5	5	5	3	1.5	1.5	32.5	-	50

94℃	2min
98℃	10s	30cycles
55℃	30s
68℃	3.5min
4℃	forever

Restriction Digestion by DpnI from 17:50 to 18:50

Electrophoresis

Name
S_ΔTMD1-E0840 (1)
S_ΔTMD1-E0840 (2)
Control

Marker: Lambda, 100bp

Ligation and Transformation

Name	Colony
S_ΔTMD1-E0840 (1)	○
S_ΔTMD1-E0840 (2)	○
Control	}

Friday, August 20 By: Wataru, Ken

Making Culture and Master Plate of S_ΔTMD1-E0840

Miniprep

Name	Concentration
B0015	41.1 ng/µL

PCR of SRRz

Name	10xBuffer	MgS04	dNTP	Template	Primer Fwd.		Primer Rev.	MilliQ	KOD plus ver.2	Total
SRRz_Sam7 (1)	5 µL	3	5	5	F1	1.5	1.5	28	1	50
SRRz_Sam7 (2)	5	3	5	5	F2	1.5	1.5	28	1	50
SRRz_Sam7 (3)	5	3	5	5	F1	1.5	1.5	28	1	50
SRRz_Sam7 (4)	5	3	5	5	F2	1.5	1.5	28	1	50
SRRz_Sam7 (5)	5	3	5	5	F1	1.5	1.5	28	1	50
SRRz_Sam7 (6)	5	3	5	5	F2	1.5	1.5	28	1	50

94℃	2min
98℃	10s	30cycles
55℃	30s
68℃	2min
4℃	forever

Electrophoresis

Name
SRRz_Sam7 (1)
SRRz_Sam7 (3)
SRRz _Sam7(5)
SRRz_Sam7 (2)
SRRz_Sam7 (4)
SRRz_Sam7 (6)

Discussion: Primer F1 might be better than F2, because the bands of 1, 2 and 3 were clearer. We decided to use sample 1 and 3. Their bands were clearer in the three.

PCR Purification

Name	Concentration
SRRz_Sam7 (1)	134.0 ng/µL
SRRz_Sam7 (3)	69.0

Restriction Digestion

Name	Sample	10xBuffer	100xBuffer	EcoRI	XbaI	SpeI	MilliQ	Total	Incubation
B0015 [EX]	50 µL	6	0.6	0.4	0.4	-	2.6	60	17:45-18:45
SRRz_Sam7 (1) [EP]	50	6	0.6	0.4	-	0.4	2.6	60
SRRz_Sam7 (3) [EP]	50	6	0.6	0.4	-	0.4	2.6	60

Purification

Name	Concentration
SRRz_Sam7 (1) [EP]	109.0 ng/µL
SRRz_Sam7 (2) [EP]	110.0
B0015	25.5

Ligation and Transformation

Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku

Miniprep

Sample number	Concentration
S_ΔTMD1-E0840 (1-1)	58.9 ng/µL
S_ΔTMD1-E0840 (2-2)	49.9

Sequence

Sample: S_ΔTMD1-E0840 (1-1). S_ΔTMD1-E0840 (2-2), I20260 [pSB4K5] Discussion: The sequencing was in success and the results were desirable. It meant the point mutation was succeeded and sequence of I20260 [pSB4K5] was confirmed. We decided to use S_ΔTMD1-E0840.

Screening PCR of SRRz_Sam7-B0015

90℃	10min
94℃	30s	35cycles
50℃	30s
72℃	1.5min
72℃	4min
4℃	hold

No.	Name
1-13	SRRz_Sam7-B0015
C	Control: B0015
N	None

Marker: Lambda, 100bp

Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.

Deletion PCR of S_ΔTMD1-E0840 (2-2)

Name	Sample	10xBuffer	dNTPs	Primer Fwd.	Primer Rev.	Template	Water	KOD-plus-
rrS_ΔTMD1-E0840 (1)	5 µL	5	1.5	1.5	1	35	1	50
rrS_ΔTMD1-E0840 (2)	5	5	1.5	1.5	1	35	1	50
rrS_ΔTMD1-E0840 (Control)	5	5	1.5	1.5	1	35	-	50

94℃	2min
94℃	10s	35cycles
56℃	30s
68℃	3.5min
4℃	forever

Restriction Digestion (DpnI)

Sample	DpnI	Total	Incubation
25 µL	1	26	19:00-20:10

Ligation

Name	Sample	Water	Ligation high	T4 Kinase	Total	Incubation
rrS_ΔTMD1-E0840 (1)	3 µL	6	5	1	15	20:15-21:15
rrS_ΔTMD1-E0840 (2)	3	6	5	1	15	20:15-21:15
rrS_ΔTMD1-E0840 (Control)	3	6	5	1	15

Transformation

Tuesday, August 24 By: Ken, Tomo, Tasuku, Takuya

Retry of deletion PCR of S_ΔTMD1-E0840

Name	Sample	10xBuffer	dNTPs	MgSO4	Primer1	Primer2	Template	Water	KOD-plus-
rrS_ΔTMD1-E0840 (1)	5 µL	5	3	1.5	1.5	1	32	1	50
rrS_ΔTMD1-E0840 (2)	5	5	3	1.5	1.5	1	32	1	50
Control	5	5	3	1.5	1.5	1	32	1	50

94℃	2min
94℃	10s	35cycles
58℃	30s
68℃	3.5min
4℃	hold

Restriction Digestion (DpnI)

14:15-15:15

Electrophoreis

Lane	Name
1	rrS_ΔTMD1-E0840 (1)
2	rrS_ΔTMD1-E0840 (3)
C	rrS_ΔTMD1-E0840 (Control)

Marker: 100bp, Lambda.

We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and Restriction Digestion were completed successfully.

Ligation

Point mutation of SRRz

Name	10x	dNTPs	MgSO4	Primer1	Primer2	Template	Water	KOD-plus-	total
SRRz_Sam7-B0015 (1)	5	5	3	1.5	1.5	1	32	1	50
SRRz_Sam7-B0015 (2)	5	5	3	1.5	1.5	1	32	1	50
SRRz_Sam7-B0015 (Control)	5	5	3	1.5	1.5	1	32	1	50

94℃	2min
98℃	10s	30cycles
55℃	30s
68℃	4min
4℃	hold

Restriction Digestion (DpnI), Electrophoresis and Ligation

We could find point mutation PCR and restriction enzyme of DpnI was done.

PCR of E0240

Sample	10xBuffer	dNTPs	MgSO4	VF2	VR	Template	Water	KOD-plus-	Total
E0240 (1)	5	5	3	1.5	1.5	1	31.5	1	50
E0240 (2)	5	5	3	1.5	1.5	1	31.5	1	50

PCR Purification

Name	Concentration
E0240 (1)	5.5 x 50 ng/µL
E0240 (2)	5.2 x 50

Restriction Digestion (EcoRI, PstI) and Gel Extraction

Name	Concentration
E0240 (1)	28.8 ng/µL
E0240 (2)	26.4

Transformation

Wednesday, August 25 By:Ken, Tomo, Kazuya, Tasuku, Takuya

Making culture and Master plate

Name	Colony
rrS_ΔTMD1-E0840 (1)	○
rrS_ΔTMD1-E0840 (2)	○
rrS_ΔTMD1-E0840 (Control)	}
SRRz_Sam7-B0015 (1)	○
SRRz_Sam7-B0015 (2)	○
SRRz_Sam7-B0015 (Control)	}

Miniprep

Name	Concentration
pSB4K5	29.0 ng/µL

Restriction Digestion

Sample name	Template	10xbuffer	100xbuffer	EcoRI	SpeI	PstI	Water	Total
pSB4K5	50	6	0.6	0.4	0.4	-	2.6	60
R0011 [pSB4K5]	10	4	0.4	-	0.3	0.3	25	40

Purification

Sample Name	Concentration
pSB4K5	18.4 ng/µL
R0011 [pSB4K5]	8.6

Ligation of E0240 and pSB4K5, Transformation

====Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka

Miniprep

Sample name	Concentration
J23116 (RPU0.7)	44.5 ng/µL

Restriction Digestion

Name	Template	10xbuffer	100xbuffer	SpeI	PstI	Water	Total
J23116 (RPU0.7)	25	4	0.4	0.3	0.3	10	40

Purification of

Name	Concentration
J23116 (RPU0.7)	49.8 ng/µL

Friday, August 27 By: Ken, Tomo, Kazuya, Fumitaka

Making master plate of E0240 [pSB4K5]

Sample Name	Concentration
rrS_ΔTMD1-E0840 (1-2)	20.9 ng/µL
SRRz-B0015 (1-1)	16.4

Restriction Digestion

Name	Template	10xbuffer	100xbuffer	XbaI	PstI	Water	Total	Incubation
rrS_ΔTMD1-E0840 (1-2)	45 µL	6	0.6	0.3	0.3	7.8	60	13:20-14:20
SRRz-B0015 (1-1)	45	6	0.6	0.3	0.3	7.8	60	13:20-14:20

Purification

rrS_ΔTMD1-E0840 (1-2)	44.7 ng/µL
SRRz-B0015 (1-1)	56.1

Ligation and Transformation

Name
R0011-rrS_ΔTMD1-E0840 (1-2)
J23116 (RPU0.7)- rrS_ΔTMD1-E0840 (1-2)
R0011-SRRz-B0015 (1-1) [pSB4K5]

Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken

Making culture and Master plate

Name	Colony	R0011-rrS_ΔTMD1-E0840	Many colonies
R0011-rrS_ΔTMD1-E0840 (Control)	Some colonies
J23116 (RPU0.7)- rrS_ΔTMD1-E0840	Many colonies
J23116 (RPU0.7)- rrS_ΔTMD1-E0840 (Control)	Many colonies
R0011-SRRz-B0015 (1-1) [pSB4K5]	No colony
R0011-SRRz-B0015 (1-1) [pSB4K5] (Control)	No colony

Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate. On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.

====Tuesday, August 31 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken

Miniprep

Name	Concentration
J23105 (RPU0.3)	48.5 ng/µL
R0011-rrS_ΔTMD1-E0840	107.3

Restriction Digestion

Gel Extraction of R0011-rrS_ΔTMD1-E0840 (Electrophoresis for 45min)

Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.

Purification of J23105 (RPU0.3) and R0011-rrS_ΔTMD1-E0840

Name	Concentration	J23105 (RPU0.3)	5.8 ng/µL
R0011-rrS_ΔTMD1-E0840	7.8

Ligation and Transformation

Insert	Vector
R0011-rrS_ΔTMD1-E0840	J23105 (RPU0.3)

Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken

Making culture and Master plate

Name	Colony
R0011-rrS_ΔTMD1-E0840-J23105 (RPU0.3)	Many colonies
R0011-rrS_ΔTMD1-E0840-J23105 (RPU0.3) (Control)	Many colonies

Screenig PCR of R0011-rrS_ΔTMD1-E0840-J23105 (RPU0.3)

Sample: 1-13
Control: Positive (B0015)
Maker: lambda, 100

Discussion: All of the sample except sample 10 might be self-ligation products of J23105 (RPU0.3).

Miniprep

SRRz-B0015 (1-1)	33.8 ng/µL
pSB4K5	56.0

Restriction Digestion of SRRz and pSB4K5

Name	Template	10xbuffer	100xbuffer	EcoRI	PstI	Water	Total	Incubation
SRRz-B0015 (1-1)	20 µL	4	0.4	0.3	0.3	15	40	13:25-14:30
pSB4K5	20	4	0.4	0.3	0.3	15	40	13:25-14:30

Purification

SRRz-B0015 (1-1)	6.5 ng/µL
pSB4K5	16.8

=Ligation and transformation

Insert: SRRz-B0015 (1-1)
Vector: pSB4K5

Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken

Making culture and Master plate

SRRz-B0015 (1-1) [pSB4K5]	13 colonies
SRRz-B0015 (1-1) [pSB4K5] (Control)	13 colonies

Screening PCR of rSRRz low

Sample: (1-13) SRRz-B0015 (1-1) [pSB4K5] Maker: Lambda, 100 Control: Positive (B0015), Neganive Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, SRRz-B0015 might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.

Friday, September 3 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken

Making culture

R0011-rrS_ΔTMD1-E0840 (1)
R0011-rrS_ΔTMD1-E0840 (3)
rrS_ΔTMD1-E0840 (1-1)
rrS_ΔTMD1-E0840 (1-2)
SRRz-B0015 (1-1) [pSB4K5]
SRRz-B0015 (1-2) [pSB4K5]
R0011-E0240 [pSB4K5]

@@ Line 3: / Line 3: @@
 ===Construction for Lysisbox===
 ====Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>====
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Colony
@@ Line 28: / Line 28: @@
 ====Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>====
 =====Culture at 37&#x2103; from 07/21 20:50 to 07/22 17:00 and Making Master Plate=====
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Colony
@@ Line 36: / Line 36: @@
 |B0015||1-23-L||1||20||21||&#x25CB;
 |}
-=====[[Team:Kyoto/Protocols#Stantard PCR|PCR]] for SRRz and S=====
+=====PCR for SRRz and S=====
 {| class="experiments"
 !No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd.||Primer Rev. (SRRz)||Primer Rev. (S)||KOD Plus ver.2||Total
@@ Line 71: / Line 71: @@
 ====Thursday, July 22 <span class="by">By: Wataru</span>====
-=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products=====
+=====Electrophoresis (40min) of the PCR Products=====
 [[Image:KyotoExp100722-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 93: / Line 93: @@
 |}
 Marker: 100bp, 1kb, 1kb, 100bp.
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 114: / Line 114: @@
 ====Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>====
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 123: / Line 123: @@
 |}
 We lost B0015 by our mistake. The concentration of pSB4K5 is high, so this condition of shaking incubation is moderate.
-=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
+=====PCR Purification=====
 {| class="experiments"
 !No.||Name||Concentration||New Name
@@ Line 136: / Line 136: @@
 |}
 The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
-=====[[Team:Kyoto/Protocols#Standard_PCR|PCR]] for SRRz=====
+=====PCR for SRRz=====
 {| class="experiments"
 !No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd. (SRRz)||Primer Rev. (SRRz)||KOD plus ver.2||Total
@@ Line 164: / Line 164: @@
 |4&#x2103;||forever||
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (35min) to check function of our Restriction Enzyme=====
+=====Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme=====
 {| class="experiments"
 !No.||Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme||MilliQ||Total||Incubation
@@ Line 181: / Line 181: @@
 Marker: 1kb.
 Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes.  So, our restriction enzymes work correctly.
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Ligation|Ligation]] to insert S gene to E0840=====
+=====Restriction Digestion and Ligation to insert S gene to E0840=====
 {| class="experiments"
 !Name||Sample||10xBuffer||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
@@ Line 202: / Line 202: @@
 ====Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>====
-=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products=====
+=====Electrophoresis of PCR Products=====
 [[Image:KyotoExp100726-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 231: / Line 231: @@
 |6||SRRZ||59.6||SRRz<sub>Sam7</sub>(2)
 |}
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Colony
@@ Line 245: / Line 245: @@
 ====Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi====
-=====[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-E0840 (Electrophoresis for 35min)=====
+=====Colony PCR of S<sub>Sam7</sub>-E0840 (Electrophoresis for 35min)=====
 [[Image:KyotoExp100727-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 281: / Line 281: @@
 |}
 Marker: 1kb, 100bp
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 291: / Line 291: @@
 |E0840||120.1
 |}
-=====[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]=====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
@@ Line 301: / Line 301: @@
 |SRRz<sub>Sam7</sub>(2)||40||5||0.5||EcoRI||0.4||SpeI||0.4||3.8||50
 |}
-=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
+=====Ligation=====
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Sample||Competent Cells||Total||Plate||Incubation||Colony
@@ Line 313: / Line 313: @@
 ====Wednesday, July 28 <span class="by">By: </span>====
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 322: / Line 322: @@
 |}
 Diluted S<sub>Sam7</sub>(1)-E0840 and S<sub>Sam7</sub>(2)-E0840 20 times with water, and used as template DNA.
-=====[[Team:Kyoto/Protocols#Mutagenesis (Point mutation, Deletion, Insertion)|Deletion PCR]] to delete a functional domain of S gene=====
+=====Deletion PCR to delete a functional domain of S gene=====
 {| class="experiments"
 !||Water||MgSO4||dNTPs||10xBuffer||Primer Fwd.||Primer Rev.||Template (1)||Template (2)||KOD Plus ver.2||Total
@@ Line 346: / Line 346: @@
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI=====
+=====Restriction Digestion to check the function of DpnI=====
 {| class="experiments"
 !Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
@@ Line 354: / Line 354: @@
 |S<sub>Sam7</sub>(2)-E0840||3||1||0.1||5.8||10
 |}
-=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min=====
+=====Electrophoresis for 35min=====
-[[image:KyotoExp100728-1.png|300px|right]]
+[[Image:KyotoExp100728-1.png|300px|right]]
 {| class="electrophoresis"
 !No.||Name||Length||Result
@@ Line 372: / Line 372: @@
 ====Thursday, July 29 <span class="by">By: </span>====
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
@@ Line 380: / Line 380: @@
 |S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (1)||50||6||DpnI||0.2||3.8||60
 |}
-=====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]=====
+=====Ligation and Phosphorylation=====
 {| class="experiments"
 !Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
@@ Line 388: / Line 388: @@
 |S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (1)||2||7||5||1||15
 |}
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Colony
@@ Line 399: / Line 399: @@
 ====Monday, August 2 <span class="by">By: Wataru, Ken</span>====
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 413: / Line 413: @@
 |R0011||18.6
 |}
-=====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of E0240=====
+=====PCR of E0240=====
 E0240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.
 {| class="experiments"
@@ Line 433: / Line 433: @@
 |4&#x2103;||forever||
 |}
-=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]=====
+=====Electrophoresis=====
-=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration
@@ Line 442: / Line 442: @@
 |E0240(2)||55.3
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] for inserting E0240 to pSB4K5 by 3A assembly=====
+=====Restriction Digestion for inserting E0240 to pSB4K5 by 3A assembly=====
 {| class="experiments"
 !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 478: / Line 478: @@
 |4&#x2103;||forever||
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] of S<sub>Sam7,&Delta;TMD1</sub>-E0840 by DpnI=====
+=====Restriction Digestion of S<sub>Sam7,&Delta;TMD1</sub>-E0840 by DpnI=====
-=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
+=====Transformation=====
 {| class="experiments"
 !Name||Sample||Competent Cells||Total||Plate||Incubation||Colony
@@ Line 493: / Line 493: @@
 =====Culture=====
 Picked two colonies from S<sub>Sam7,&Delta;TMD1</sub>-E0840 (1), and S<sub>Sam7,&Delta;TMD1</sub>-E0840 (3), and cultured at 37&#x2103; from 08/03 to 08/04.
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
+=====Miniprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 501: / Line 501: @@
 |R0011||26.8
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 526: / Line 526: @@
 =====Ethanol Precipitation=====
 After ethanol precipitation, we diluted pSB4K5 by 2&micro;L MilliQ
-=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
+=====Ligation=====
 {| class="experiments"
 !Name||Vector||colspan="2"|Insert 1||colspan="2"|Insert 2||Ligation High||Total||Incubation
@@ Line 534: / Line 534: @@
 |R0011-E0240(2) [pSB4K5]||pSB4K5 [EP]||1||R0011 [ES]||1||E0240(2) [XP]||1||3||15
 |}
-=====[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of I20260=====
+=====PCR of I20260=====
 {| class="experiments"
 !Name||Water||MgSO4||dNTPs||10xBuffer||Primer VF2||Primer VR||Template I20260||KOD plus ver.2||Total
@@ Line 553: / Line 553: @@
 |4&#x2103;||forever||
 |}
-=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration
@@ Line 559: / Line 559: @@
 |I20260||40.6 ng/&micro;L
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 565: / Line 565: @@
 |I20260 [EP]||45 &micro;L||6||0.6||EcoRI||0.2||PstI||0.2||8||60
 |}
-=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration||Volume
@@ Line 572: / Line 572: @@
 |}
 I20260 [EP] is concentrated at 7&micro;L
-=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
+=====Ligation=====
 {| class="experiments"
 !||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
@@ Line 721: / Line 721: @@
 |13||I20260 [pSB4K5] (2) [EP]||960, 4339||&#x25CB;
 |}
-[[Image:KyotoExp100806-1.png]]
+[[Image:KyotoExp100806-1.png|300px|right]]
 White colonies are not inserted R0011 but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of ''lac''I gene.
 =====Error PCR (Retry)=====
@@ Line 885: / Line 885: @@
 |8||R0011 [pSB4K5, C2] (2-N)||||
 |}
-[[image:KyotoExp100811-1.png]]
+[[image:KyotoExp100811-1.png|300px|right]]
 Each enzyme correctly cut samples.
 =====Screening PCR of SRRz=====
@@ Line 902: / Line 902: @@
 |}
 Marker: Lambda Marker
-[[image:KyotoExp100811-2.png]]
+[[image:KyotoExp100811-2.png|300px|right]]
-[[image:KyotoExp100811-3.png]]
+[[image:KyotoExp100811-3.png|300px|right]]
 Discussion: All of the sample were self-ligation of DT. SRRz weren't inserted.
@@ Line 927: / Line 927: @@
 |}
 Maker: Lambda, 100bp
-[[image:KyotoExp100812-1.png]]
+[[image:KyotoExp100812-1.png|300px|right]]
 Discussion: Each enzyme correctly cut each sample and was active.
@@ Line 971: / Line 971: @@
 |}
 Marker: Lambda, 100bp
-[[Image:KyotoExp100819-1.png]]
+[[Image:KyotoExp100819-1.png|300px|right]]
 =====Ligation and Transformation=====
 {| class="experiments
@@ Line 986: / Line 986: @@
 ====Friday, August 20 <span class="by">By: Wataru, Ken</span>====
 =====Making Culture and Master Plate of S<sub>&Delta;TMD1</sub>-E0840=====
-=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]=====
+=====Miniprep=====
 {| class="expeirments"
 !Name||Concentration
@@ Line 992: / Line 992: @@
 |B0015||41.1 ng/&micro;L
 |}
-=====[[Team:Kyoto/Protocols#Standard PCR|PCR]] of SRRz=====
+=====PCR of SRRz=====
 {| class="experiments"
 !Name||10xBuffer||MgS04||dNTP||Template||colspan="2"|Primer Fwd.||Primer Rev.||MilliQ||KOD plus ver.2||Total
 |-
-|SRRz (1)||5 &micro;L||3||5||5||F1||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (1)||5 &micro;L||3||5||5||F1||1.5||1.5||28||1||50
 |-
-|SRRz (2)||5||3||5||5||F2||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (2)||5||3||5||5||F2||1.5||1.5||28||1||50
 |-
-|SRRz (3)||5||3||5||5||F1||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (3)||5||3||5||5||F1||1.5||1.5||28||1||50
 |-
-|SRRz (4)||5||3||5||5||F2||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (4)||5||3||5||5||F2||1.5||1.5||28||1||50
 |-
-|SRRz (5)||5||3||5||5||F1||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (5)||5||3||5||5||F1||1.5||1.5||28||1||50
 |-
-|SRRz (6)||5||3||5||5||F2||1.5||1.5||28||1||50
+|SRRz<sub>Sam7</sub> (6)||5||3||5||5||F2||1.5||1.5||28||1||50
 |}
 {| class="experiments"
@@ Line 1,019: / Line 1,019: @@
 |4&#x2103;||forever||
 |}
-=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]=====
+=====Electrophoresis=====
 {| class="electrophoresis"
 !Name
 |-
-|SRRz (1)
+|SRRz<sub>Sam7</sub> (1)
 |-
-|SRRz (3)
+|SRRz<sub>Sam7</sub> (3)
 |-
-|SRRz (5)
+|SRRz <sub>Sam7</sub>(5)
 |-
-|SRRz (2)
+|SRRz<sub>Sam7</sub> (2)
 |-
-|SRRz (4)
+|SRRz<sub>Sam7</sub> (4)
 |-
-|SRRz (6)
+|SRRz<sub>Sam7</sub> (6)
 |}
-[[Image:KyotoExp100820-1.png]]
+[[Image:KyotoExp100820-1.png|300px|right]]
 Discussion: Primer F1 might be better than F2, because the bands of 1, 2 and 3 were clearer. We decided to use sample 1 and 3. Their bands were clearer in the three.
-=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration
 |-
-|SRRz (1)||134.0 ng/&micro;L
+|SRRz<sub>Sam7</sub> (1)||134.0 ng/&micro;L
 |-
-|SRRz (3)||69.0
+|SRRz<sub>Sam7</sub> (3)||69.0
 |}
-=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample||10xBuffer||100xBuffer||EcoRI||XbaI||SpeI||MilliQ||Total||Incubation
@@ Line 1,051: / Line 1,051: @@
 |B0015 [EX]||50 &micro;L||6||0.6||0.4||0.4||-||2.6||60||rowspan="3"|17:45-18:45
 |-
-|SRRz (1) [EP]||50||6||0.6||0.4||-||0.4||2.6||60
+|SRRz<sub>Sam7</sub> (1) [EP]||50||6||0.6||0.4||-||0.4||2.6||60
 |-
-|SRRz (3) [EP]||50||6||0.6||0.4||-||0.4||2.6||60
+|SRRz<sub>Sam7</sub> (3) [EP]||50||6||0.6||0.4||-||0.4||2.6||60
 |}
 =====Purification=====
@@ Line 1,059: / Line 1,059: @@
 !Name||Concentration
 |-
-|SRRz (1) [EP]||109.0 ng/&micro;L
+|SRRz<sub>Sam7</sub> (1) [EP]||109.0 ng/&micro;L
 |-
-|SRRz (2) [EP]||110.0
+|SRRz<sub>Sam7</sub> (2) [EP]||110.0
 |-
 |B0015||25.5
 |}
-=====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Transformation]]=====
+=====Ligation and Transformation=====
+----
+====Monday, August 23 <span class="by">By: Wataru, Tomo, Ken, Fumitaka, Tasuku</span>====
+=====Miniprep=====
+{| class="experiments"
+|Sample number||Concentration
+|-
+|S<sub>&Delta;TMD1</sub>-E0840 (1-1)||58.9 ng/&micro;L
+|-
+|S<sub>&Delta;TMD1</sub>-E0840 (2-2)||49.9
+|}
+=====Sequence=====
+Sample: S<sub>&Delta;TMD1</sub>-E0840 (1-1). S<sub>&Delta;TMD1</sub>-E0840 (2-2), I20260 [pSB4K5]
+Discussion: The sequencing was in success and the results were desirable. It meant the point mutation was succeeded and sequence of I20260 [pSB4K5] was confirmed. We decided to use S<sub>&Delta;TMD1</sub>-E0840.
+=====Screening PCR of SRRz<sub>Sam7</sub>-B0015=====
+{| class="experiments"
+|90&#x2103;||10min||
+|-
+|94&#x2103;||30s||rowspan="3"|35cycles
+|-
+|50&#x2103;||30s
+|-
+|72&#x2103;||1.5min
+|-
+|72&#x2103;||4min||
+|-
+|4&#x2103;||hold||
+|}
+{| class="electrophoresis"
+!No.||Name
+|-
+|1-13||SRRz<sub>Sam7</sub>-B0015
+|-
+|C||Control: B0015
+|-
+|N||None
+|}
+Marker: Lambda, 100bp
+[[Image:KyotoExp100823-1.png|300px|right]]
+Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
+=====Deletion PCR of S<sub>&Delta;TMD1</sub>-E0840 (2-2)=====
+{| class="experiments"
+!Name||Sample||10xBuffer||dNTPs||Primer Fwd.||Primer Rev.||Template||Water||KOD-plus-||Total
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1)||5 &micro;L||5||1.5||1.5||1||35||1||50
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (2)||5||5||1.5||1.5||1||35||1||50
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (Control)||5||5||1.5||1.5||1||35||-||50
+|}
+{| class="experiments"
+|94&#x2103;||2min||
+|-
+|94&#x2103;||10s||rowspan="3"|35cycles
+|-
+|56&#x2103;||30s
+|-
+|68&#x2103;||3.5min
+|-
+|4&#x2103;||forever||
+|}
+=====Restriction Digestion (DpnI)=====
+{| class="experiments"
+!Sample||DpnI||Total||Incubation
+|-
+|25 &micro;L||1||26||19:00-20:10
+|}
+=====Ligation=====
+{| class="experiments"
+!Name||Sample||Water||Ligation high||T4 Kinase||Total||Incubation
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1)||3 &micro;L||6||5||1||15||20:15-21:15
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (2)||3||6||5||1||15||20:15-21:15
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (Control)||3||6||5||1||15
+|}
+=====Transformation=====
+====Tuesday, August 24 <span class="by">By: Ken, Tomo, Tasuku, Takuya</span>====
+=====Retry of deletion PCR of S<sub>&Delta;TMD1</sub>-E0840=====
+{| class="experiments"
+!Name||Sample||10xBuffer||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||Total
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840  (1)||5 &micro;L||5||3||1.5||1.5||1||32||1||50
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840  (2)||5||5||3||1.5||1.5||1||32||1||50
+|-
+|Control||5||5||3||1.5||1.5||1||32||1||50
+|}
+{| class="experiments"
+|94&#x2103;||2min||
+|-
+|94&#x2103;||10s||rowspan="3"|35cycles
+|-
+|58&#x2103;||30s
+|-
+|68&#x2103;||3.5min
+|-
+|4&#x2103;||hold||
+|}
+=====Restriction Digestion (DpnI)=====
+:15-15:15
+=====Electrophoreis=====
+{| class="electrophoresis"
+!Lane||Name
+|-
+|1||rrS<sub>&Delta;TMD1</sub>-E0840 (1)
+|-
+|2||rrS<sub>&Delta;TMD1</sub>-E0840 (3)
+|-
+|C||rrS<sub>&Delta;TMD1</sub>-E0840 (Control)
+|}
+Marker: 100bp, Lambda.
+[[Image:KyotoExp100824-1.png|300px|right]]
+We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and Restriction Digestion were completed successfully.
+=====Ligation=====
+=====Point mutation of SRRz=====
+{| class="experiments"
+!Name||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||total
+|-
+|SRRz<sub>Sam7</sub>-B0015 (1)||5||5||3||1.5||1.5||1||32||1||50
+|-
+|SRRz<sub>Sam7</sub>-B0015 (2)||5||5||3||1.5||1.5||1||32||1||50
+|-
+|SRRz<sub>Sam7</sub>-B0015 (Control)||5||5||3||1.5||1.5||1||32||1||50
+|}
+{| class="experiments"
+|94&#x2103;||2min||
+|-
+|98&#x2103;||10s||rowspan="3"|30cycles
+|-
+|55&#x2103;||30s
+|-
+|68&#x2103;||4min
+|-
+|4&#x2103;||hold||
+|}
+=====Restriction Digestion (DpnI), Electrophoresis and Ligation=====
+[[Image:KyotoExp100824-2.png|300px|right]]
+We could find point mutation PCR and restriction enzyme of DpnI was done.
+====PCR of E0240====
+{| class="experiments"
+!Sample||10xBuffer||dNTPs||MgSO4||VF2||VR||Template||Water||KOD-plus-||Total
+|-
+|E0240 (1)||5||5||3||1.5||1.5||1||31.5||1||50
+|-
+|E0240 (2)||5||5||3||1.5||1.5||1||31.5||1||50
+|-
+|}
+=====PCR Purification=====
+{| class="experiments"
+!Name||Concentration
+|-
+|E0240 (1)||5.5 x 50 ng/&micro;L
+|-
+|E0240 (2)||5.2 x 50
+|}
+=====Restriction Digestion (EcoRI, PstI) and Gel Extraction=====
+{| class="experiments"
+!Name||Concentration
+|-
+|E0240 (1)|| 28.8 ng/&micro;L
+|-
+|E0240 (2)|| 26.4
+|}
+=====Transformation=====
+====Wednesday, August 25 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya<span>====
+=====Making culture and Master plate=====
+{| class="experiments"
+!Name||Colony
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1)||&#x25CB;
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (2)||&#x25CB;
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (Control)||&#x7D;
+|-
+|SRRz<sub>Sam7</sub>-B0015 (1)||&#x25CB;
+|-
+|SRRz<sub>Sam7</sub>-B0015 (2)||&#x25CB;
+|-
+|SRRz<sub>Sam7</sub>-B0015 (Control)||&#x7D;
+|}
+=====Miniprep=====
+{| class="experiments"
+!Name||Concentration
+|-
+|pSB4K5||29.0 ng/&micro;L
+|}
+=====Restriction Digestion=====
+{| class="experiments"
+!Sample name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||PstI||Water||Total
+|-
+|pSB4K5||50||6||0.6||0.4||0.4||-||2.6||60
+|-
+|R0011 [pSB4K5]||10||4||0.4||-||0.3||0.3||25||40
+|}
+=====Purification=====
+{| class="experiments"
+|Sample Name||Concentration
+|-
+|pSB4K5||18.4 ng/&micro;L
+|-
+|R0011 [pSB4K5]||8.6
+|}
+=====Ligation of E0240 and pSB4K5, Transformation=====
+====Thursday, August 26 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka</span>
+=====Miniprep=====
+{| class="experiments"
+|Sample name||Concentration
+|-
+|J23116 (RPU0.7)||44.5 ng/&micro;L
+|}
+=====Restriction Digestion=====
+{| class="experiments"
+!Name||Template||10xbuffer||100xbuffer||SpeI||PstI||Water||Total
+|-
+|J23116 (RPU0.7)||25||4||0.4||0.3||0.3||10||40
+|}
+=====Purification of=====
+{| class="experiments"
+!Name||Concentration
+|-
+|J23116 (RPU0.7)||49.8 ng/&micro;L
+|}
+====Friday, August 27 <span class="by">By: Ken, Tomo, Kazuya, Fumitaka</span>====
+=====Making master plate of E0240 [pSB4K5]=====
+{| class="experiments"
+|Sample Name||Concentration
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)||20.9 ng/&micro;L
+|-
+|SRRz-B0015 (1-1)||16.4
+|}
+=====Restriction Digestion=====
+{| class="experiments"
+!Name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total||Incubation
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)||45 &micro;L||6||0.6||0.3||0.3||7.8||60||rowspan="2"|13:20-14:20
+|-
+|SRRz-B0015 (1-1)||45||6||0.6||0.3||0.3||7.8||60
+|}
+=====Purification=====
+{|Sample Name||Concentration
+|-
+|rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)||44.7 ng/&micro;L
+|-
+|SRRz-B0015 (1-1)||56.1
+|}
+=====Ligation and Transformation=====
+{| class="experiments"
+!Name
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)
+|-
+|J23116 (RPU0.7)- rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)
+|-
+|R0011-SRRz-B0015 (1-1) [pSB4K5]
+|}
+====Monday, August 30 <span class="by">By: Tomonori, Kazuya, Tasuku, Ken</span>====
+=====Making culture and Master plate=====
+{| class="experiments"
+!Name||Colony
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840||Many colonies
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840 (Control)||Some colonies
+|-
+|J23116 (RPU0.7)- rrS<sub>&Delta;TMD1</sub>-E0840||Many colonies
+|-
+|J23116 (RPU0.7)- rrS<sub>&Delta;TMD1</sub>-E0840 (Control)||Many colonies
+|-
+|R0011-SRRz-B0015 (1-1) [pSB4K5]||No colony
+|-
+|R0011-SRRz-B0015 (1-1) [pSB4K5] (Control)||No colony
+|}
+Discussion: There ware some colonies, which emitted green light, on the plate 1.  So, we cultured those colonies on master plate.
+On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.
+====Tuesday, August 31 <span class="by">By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken<span>
+=====Miniprep=====
+{| class="experiments"
+!Name||Concentration
+|-
+|J23105 (RPU0.3)||48.5 ng/&micro;L
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840||107.3
+|}
+=====Restriction Digestion=====
+=====Gel Extraction of R0011-rrS<sub>&Delta;TMD1</sub>-E0840 (Electrophoresis for 45min)=====
+<!--[[image:KyotoExp100831-1.png|300px|right]]-->
+Discussion: There were two band at the bottom of the gel.  It was too long -45min-, and insert and vector might be contaminated.  But we went on next operation.
+=====Purification of J23105 (RPU0.3) and R0011-rrS<sub>&Delta;TMD1</sub>-E0840=====
+{| class="experiments"
+!Name||Concentration
+|J23105 (RPU0.3)||5.8 ng/&micro;L
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840||7.8
+|}
+=====Ligation and Transformation=====
+{| class="experiments"
+|Insert||Vector
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840||J23105 (RPU0.3)
+|}
+====Wednesday, September 1 <span class="by">By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken</span>====
+=====Making culture and Master plate=====
+{| class="experiments"
+!Name||Colony
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840-J23105 (RPU0.3)||Many colonies
+|-
+|R0011-rrS<sub>&Delta;TMD1</sub>-E0840-J23105 (RPU0.3) (Control)||Many colonies
+|}
+=====Screenig PCR of R0011-rrS<sub>&Delta;TMD1</sub>-E0840-J23105 (RPU0.3)=====
+* Sample: 1-13
+* Control: Positive (B0015)
+* Maker: lambda, 100
+<!--M, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, P, M
+[[image:KyotoExp100901-1.png|300px|right]]-->
+Discussion: All of the sample except sample 10 might be self-ligation products of J23105 (RPU0.3).
+=====Miniprep=====
+{| class="experiments"
+|SRRz-B0015 (1-1)||33.8 ng/&micro;L
+|-
+|pSB4K5||56.0
+|}
+=====Restriction Digestion of SRRz and pSB4K5=====
+{| class="experiments"
+!Name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total||Incubation
+|-
+|SRRz-B0015 (1-1)||20 &micro;L||4||0.4||0.3||0.3||15||40||rowspan="2"|13:25-14:30
+|-
+|pSB4K5||20||4||0.4||0.3||0.3||15||40
+|}
+=====Purification=====
+{| class="experiments"
+|SRRz-B0015 (1-1)||6.5 ng/&micro;L
+|-
+|pSB4K5||16.8
+|}
+=====Ligation and transformation====
+* Insert: SRRz-B0015 (1-1)
+* Vector: pSB4K5
+====Thursday, September 2 <span class="by">By: Tomonori, Tomo, Takuya, Ken<span>====
+=====Making culture and Master plate=====
+{| class="experiments"
+|SRRz-B0015 (1-1) [pSB4K5]||13 colonies
+|-
+|SRRz-B0015 (1-1) [pSB4K5] (Control)||13 colonies
+|}
+=====Screening PCR of rSRRz low=====
+Sample: (1-13) SRRz-B0015 (1-1) [pSB4K5]
+Maker: Lambda, 100
+Control: Positive (B0015), Neganive
+<!--M, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, P, N, M
+[[image:KyotoExp100902-1.png|300px|right]]-->
+Discussion: From sample 1, two vectors might be ligated.  Sample 3 and 4, SRRz-B0015 might be inserted in low copy plasmid correctly.  Sample 11, it might be the self-ligation product of low copy plasmid.  Anyway, we decided to culture those 4 colonies on master plate.
+====Friday, September 3 <span class="by">By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken</span>====
+=====Making culture=====
+*R0011-rrS<sub>&Delta;TMD1</sub>-E0840 (1)
+*R0011-rrS<sub>&Delta;TMD1</sub>-E0840 (3)
+*rrS<sub>&Delta;TMD1</sub>-E0840 (1-1)
+*rrS<sub>&Delta;TMD1</sub>-E0840 (1-2)
+*SRRz-B0015 (1-1) [pSB4K5]
+*SRRz-B0015 (1-2) [pSB4K5]
+*R0011-E0240 [pSB4K5]
 ----

Name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Team:Kyoto/Notebook1

From 2010.igem.org

Revision as of 14:02, 23 October 2010

Contents

Notebook

Construction for Lysisbox

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Transformation

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate

Transformation

PCR for SRRz and S

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

Miniprep

Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of pSB4K5 and B0015

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

PCR Purification

PCR for SRRz

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme

Restriction Digestion and Ligation to insert S gene to E0840

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

PCR Purification

Transformation

Culture of pSB4K5, E0840, and B0015

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Colony PCR of SSam7-E0840 (Electrophoresis for 35min)

Miniprep

Restriction Digestion

Ligation

Transformation

Wednesday, July 28 By:

Miniprep

Deletion PCR to delete a functional domain of S gene

Restriction Digestion to check the function of DpnI

Electrophoresis for 35min

Thursday, July 29 By:

Restriction Digestion

Ligation and Phosphorylation

Transformation

Monday, August 2 By: Wataru, Ken

Miniprep

PCR of E0240

Electrophoresis

PCR Purification

Restriction Digestion for inserting E0240 to pSB4K5 by 3A assembly

PCR Purification

Error PCR

Restriction Digestion of SSam7,ΔTMD1-E0840 by DpnI

Transformation

Tuesday, August 3 By:

Culture

Miniprep

Restriction Digestion

PCR Purification

Ethanol Precipitation

Ligation

PCR of I20260

PCR Purification

Restriction Digestion

PCR Purification

Ligation

Transformation

Thursday, August 5 By:

Culture and Master Plates

Sequence

Friday, August 6

Miniprep

Restriction Digestion

==Electrophoresis

Error PCR (Retry)

Transformation

Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep

Transfotrmation

Restriction Eigestion and Ethanol Precipitation

Ligation and Transformation

Culture

Colony PCR of S_Sam7-E0840 (Electrophoresis for 35min)

Restriction Digestion of S_Sam7,ΔTMD1-E0840 by DpnI

Miniprep of S_Sam7,ΔTMD1-E0840

Point mutation PCR of S_Sam7,ΔTMD1-E0840

Making Culture and Master Plate of S_ΔTMD1-E0840

Screening PCR of SRRz_Sam7-B0015

Deletion PCR of S_ΔTMD1-E0840 (2-2)

Retry of deletion PCR of S_ΔTMD1-E0840

Gel Extraction of R0011-rrS_ΔTMD1-E0840 (Electrophoresis for 45min)

Purification of J23105 (RPU0.3) and R0011-rrS_ΔTMD1-E0840

Screenig PCR of R0011-rrS_ΔTMD1-E0840-J23105 (RPU0.3)

Name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10