Team:Kyoto/Notebook1

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Revision as of 11:52, 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
<partinfo>J23100</partinfo>	1-18-C	1 µL	20	21	LB (Amp+)	At 37℃, 7/20 20:50 - 7/21 17:00	○
<partinfo>J23105</partinfo>	1-18-M	1	20	21			○
<partinfo>J23116</partinfo>	1-20-M	1	20	21			○
<partinfo>R0011</partinfo>	1-6-G	1	20	21			○
<partinfo>E0840</partinfo>	1-12-O	1	20	21			○
<partinfo>J06702</partinfo>	2-8-E	1	20	21			○
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21			×
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kan+)		×

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

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
<partinfo>pSB4K5</partinfo>	1-5-G	1 µL	20	21	LB (Kan+)	At 37℃, 7/21 20:50 - 7/22 16:30	○
<partinfo>B0015</partinfo>	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
<partinfo>J23100</partinfo>	18.5 (ng/µL)
<partinfo>J23105</partinfo>	12.5
<partinfo>J23116</partinfo>	14.6
<partinfo>R0011</partinfo>	8.6
<partinfo>E0840</partinfo>	12.1
<partinfo>J06702</partinfo>	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 <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

Name	Concentration
<partinfo>pSB4K5</partinfo>	79.2 (ng/µL)
<partinfo>B0015</partinfo>	-

We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> 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	<partinfo>J06702</partinfo>	5 µL	1	0.1	EcoRI	0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	<partinfo>J06702</partinfo>	5	1	0.1	XbaI	0.1	3.6	10
3	<partinfo>J06702</partinfo>	5	1	0.1	SpeI	0.1	3.6	10
4	<partinfo>J06702</partinfo>	5	1	0.1	PstI	0.1	3.6	10
5	<partinfo>J06702</partinfo>	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 <partinfo>E0840</partinfo>

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
<partinfo>E0840</partinfo>	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)-<partinfo>E0840</partinfo>	<partinfo>E0840</partinfo>	0.5µL	S_Sam7(1)	0.5	1	2
S_Sam7(2)-<partinfo>E0840</partinfo>	<partinfo>E0840</partinfo>	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
<partinfo>E0240</partinfo>	1-12-M	1 µL	20	21	LB (Amp+)	At 37℃ 7/26 - 7/27	×
<partinfo>I20260</partinfo>	2-17-F	1	20	21	LB (Kan+)		×
<partinfo>J04450</partinfo>	1-5-E	1	20	21	LB (Kan+)		×

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>

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

Colony PCR of S_Sam7-<partinfo>E0840</partinfo> (Electrophoresis for 35min)

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

Marker: 1kb, 100bp

Miniprep

Name	Concentration
<partinfo>R0011</partinfo>	26.9 ng/µL
<partinfo>B0015</partinfo>	120.0
<partinfo>E0840</partinfo>	120.1

Restriction Digestion

Name	Sample	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
<partinfo>B0015</partinfo>	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)-<partinfo>E0840</partinfo>	95.5 ng/µL
S_Sam7(2)-<partinfo>E0840</partinfo>	98.6

Diluted S_Sam7(1)-<partinfo>E0840</partinfo> and S_Sam7(2)-<partinfo>E0840</partinfo> 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)-<partinfo>E0840</partinfo> (1)	28 µL	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(1)-<partinfo>E0840</partinfo> (2)	28	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(2)-<partinfo>E0840</partinfo> (1)	28	3	5	5	1.5	1.5	-	5	1	50
S_Sam7,ΔTMD1(2)-<partinfo>E0840</partinfo> (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)-<partinfo>E0840</partinfo>	3 µL	1	0.1	5.8	10
S_Sam7(2)-<partinfo>E0840</partinfo>	3	1	0.1	5.8	10

Electrophoresis for 35min

No.	Name	Length
1	Not digested S_Sam7(1)-<partinfo>E0840</partinfo>	3363bp
2	Not digested S_Sam7(2)-<partinfo>E0840</partinfo>	3363
3	Digested S_Sam7(1)-<partinfo>E0840</partinfo>	1021, 933, 402, 341, 258, 105, ...
4	Digested S_Sam7(2)-<partinfo>E0840</partinfo>	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)-<partinfo>E0840</partinfo> (1)	50 µL	6	DpnI	0.2	3.8	60	07/29 09:40 - 07/29 11:00
S_Sam7,ΔTMD1(2)-<partinfo>E0840</partinfo> (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)-<partinfo>E0840</partinfo> (1)	2 µL	7	5	1	15	07/29 11:30 ~ 07/29 13:00
S_Sam7,ΔTMD1(2)-<partinfo>E0840</partinfo> (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)-<partinfo>E0840</partinfo> (1)	3 µL	30	33	LB (Amp+)	07/29 ~ 07/30	○
S_Sam7,ΔTMD1(2)-<partinfo>E0840</partinfo> (1)	3	30	33	LB (Amp+)	07/29 ~ 07/30	○

Monday, August 2 By: Wataru, Ken

Miniprep

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

Standard PCR of <partinfo>E0240</partinfo>

<partinfo>E0240</partinfo> 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 <partinfo>E0240</partinfo>	KOD Pllus ver.2	Total
<partinfo>E0240</partinfo>(1)	28 µL	3	5	5	1.5	1.5	5	1	50
<partinfo>E0240</partinfo>(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
<partinfo>E0240</partinfo>(1)	42.6 ng/µL
<partinfo>E0240</partinfo>(2)	55.3

Restriction Digestion for inserting <partinfo>E0240</partinfo> to <partinfo>pSB4K5</partinfo> by 3A assembly

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

PCR Purification

Name	Concentration	Volume
<partinfo>E0240</partinfo>(1) [XP]	21.8 ng/µL	40 µL
<partinfo>E0240</partinfo>(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-<partinfo>E0840</partinfo> (1)	32 µL	3	5	5	1.5	1.5	1	-	-	1	50
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (2)	32	3	5	5	1.5	1.5	-	1	-	1	50
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (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-<partinfo>E0840</partinfo> by DpnI

Transformation

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

Tuesday, August 3 By:

Culture

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

Miniprep

Name	Concentration
<partinfo>pSB4K5</partinfo>	60.7 ng/µL
<partinfo>R0011</partinfo>	26.8

Restriction Digestion

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

PCR Purification

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

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

Ethanol Precipitation

After ethanol precipitation, we diluted <partinfo>pSB4K5</partinfo> by 2µL MilliQ

Ligation

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

Standard PCR of <partinfo>I20260</partinfo>

Name	Water	MgSO4	dNTPs	10xBuffer	Primer VF2	Primer VR	Template <partinfo>I20260</partinfo>	KOD plus ver.2	Total
<partinfo>I20260</partinfo> (1)	32µL	3	5	5	1.5	1.5	1	1	50
<partinfo>I20260</partinfo> (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
<partinfo>I20260</partinfo>	40.6 ng/µL

Restriction Digestion

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

PCR Purification

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

<partinfo>I20260</partinfo> [EP] is concentrated at 7µL

Ligation

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

Transformation

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

Thursday, August 5 By:

Culture and Master Plates

<partinfo>pSB4K5</partinfo> 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 <partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>] and <partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>] plate. We cultured both white and green colonies.

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

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

Sequence

Name	Concentration
S_ΔTMD1-<partinfo>E0840</partinfo>(1) A	28.9 ng/µL
S_ΔTMD1-<partinfo>E0840</partinfo>(1) B	25.3
S_ΔTMD1-<partinfo>E0840</partinfo>(3) A	26.6
S_ΔTMD1-<partinfo>E0840</partinfo>(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
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>] (1)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>] (2)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>] (3)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>] (4)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>] (1)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>] (2)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>] (3)
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>] (4)
<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (1)
<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (2)
<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (3)

Restriction Digestion

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

==Electrophoresis

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

White colonies are not inserted <partinfo>R0011</partinfo> 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-<partinfo>E0840</partinfo> failed (50ng/µL)	KOD plus ver.2	Total
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (1)	32	3	5	5	1.5	1.5	1	1	50
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (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-<partinfo>E0840</partinfo> (1)	-	4 µL	50	54	LB (Kan+)	08/06-08/09	○
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (2)	-	4	50	54			○
<partinfo>I20260</partinfo>	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
<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>]	116.2 ng/µL
<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo> [<partinfo>pSB4K5</partinfo>]	146.6

Transfotrmation

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

Restriction Eigestion and Ethanol Precipitation

To use <partinfo>R0011</partinfo> for next ligation, we digested it by EcoRI and PstI

Name	Sample	10x Buffer	BSA	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
<partinfo>R0011</partinfo> [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
<partinfo>R0011</partinfo> [<partinfo>pSB4K5</partinfo>, KRX]	2 µL	KRX	50	52	LB (Kan+)	08/09 20:00-08/10 09:00	○
<partinfo>R0011</partinfo> [<partinfo>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 <partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>, <partinfo>R0011</partinfo>-<partinfo>E0240</partinfo> [<partinfo>pSB4K5</partinfo>], <partinfo>R0011</partinfo> [<partinfo>pSB4K5</partinfo>, KRX], and <partinfo>R0011</partinfo> [<partinfo>pSB4K5</partrinfo>, C2].

Minprep

Name	Concentration
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (1-1)	9.9 ng/µL
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (1-2)	27.3
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (2-1)	43.2
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (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 <partinfo>R0011</partinfo> [pSB4K5, C2], SRRz 1', 3'

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

Restriction Digestion and electrophoresis of <partinfo>R0011</partinfo> [pSB4K5, C2]

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

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

Each enzyme correctly cut samples.

Screening PCR of SRRz

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

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 <partinfo>B0015</partinfo>

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-<partinfo>E0840</partinfo>

Name	Concentration
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo>	29.6 ng/µL

Point mutation PCR of S_Sam7,ΔTMD1-<partinfo>E0840</partinfo>

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

Marker: Lambda, 100bp

Ligation and Transformation

Name	Colony
S_ΔTMD1-<partinfo>E0840</partinfo> (1)	○
S_ΔTMD1-<partinfo>E0840</partinfo> (2)	○
Control	}

Friday, August 20 By: Wataru, Ken

Making Culture and Master Plate of S_ΔTMD1-<partinfo>E0840</partinfo>

[[Team:Kyoto/Protocols#Miniprep|Miniprep]

Name	Concentration
<partinfo>B0015</partinfo>	41.1 ng/µL

PCR of SRRz

Name	10xBuffer	MgS04	dNTP	Template	Primer Fwd.		Primer Rev.	MilliQ	KOD plus ver.2	Total
SRRz (1)	5 µL	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 (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 (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

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

Electrophoresis

Name
SRRz (1)
SRRz (3)
SRRz (5)
SRRz (2)
SRRz (4)
SRRz (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 (1)	134.0 ng/µL
SRRz (3)	69.0

Restriction Digestion

Name	Sample	10xBuffer	100xBuffer	EcoRI	XbaI	SpeI	MilliQ	Total	Incubation
<partinfo>B0015</partinfo> [EX]	50 µL	6	0.6	0.4	0.4	-	2.6	60	17:45-18:45
SRRz (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

Purification

Name	Concentration
SRRz (1) [EP]	109.0 ng/µL
SRRz (2) [EP]	110.0
<partinfo>B0015</partinfo>	25.5

@@ Line 1: / Line 1: @@
 {{:Team:Kyoto/Header}}
-==Notebook: Construction for Lysisbox==
+==Notebook==
-===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
+===Construction for Lysisbox===
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+====Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
+!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Colony
 |-
 |<partinfo>J23100</partinfo>||1-18-C||1 &micro;L||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103;, 7/20 20:50 - 7/21 17:00||&#x25CB;
@@ Line 25: / Line 26: @@
-===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
+====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====
+=====Culture at 37&#x2103; from 07/21 20:50 to 07/22 17:00 and Making Master Plate=====
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
+!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Colony
 |-
 |<partinfo>pSB4K5</partinfo>||1-5-G||1 &micro;L||20||21||rowspan="2"|LB (Kan+)||rowspan="2"|At 37&#x2103;, 7/21 20:50 - 7/22 16:30||&#x25CB;
@@ Line 35: / Line 36: @@
 |<partinfo>B0015</partinfo>||1-23-L||1||20||21||&#x25CB;
 |}
-====[[Team:Kyoto/Protocols#Stantard PCR|PCR]] for SRRz and S====
+=====[[Team:Kyoto/Protocols#Stantard PCR|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 69: / Line 70: @@
-===Thursday, July 22 <span class="by">By: Wataru</span>===
+====Thursday, July 22 <span class="by">By: Wataru</span>====
-====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products====
+=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products=====
 [[Image:KyotoExp100722-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 92: / Line 93: @@
 |}
 Marker: 100bp, 1kb, 1kb, 100bp.
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 109: / Line 110: @@
 |}
 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 <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>====
+=====Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>=====
-===Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
+====Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>====
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 122: / Line 123: @@
 |}
 We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
-====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
 {| class="experiments"
 !No.||Name||Concentration||New Name
@@ Line 135: / 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====
+=====[[Team:Kyoto/Protocols#Standard_PCR|PCR]] for SRRz=====
 {| class="experiments"
 !No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd. (SRRz)||Primer Rev. (SRRz)||KOD plus ver.2||Total
@@ Line 163: / 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====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (35min) to check function of our Restriction Enzyme=====
 {| class="experiments"
 !No.||Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme||MilliQ||Total||Incubation
@@ Line 180: / 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 <partinfo>E0840</partinfo>====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Ligation|Ligation]] to insert S gene to <partinfo>E0840</partinfo>=====
 {| class="experiments"
 !Name||Sample||10xBuffer||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
@@ Line 200: / Line 201: @@
-===Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
+====Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>====
-====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products====
+=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products=====
 [[Image:KyotoExp100726-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 220: / Line 221: @@
 Marker: 1kb.
 At the condition 4 (4.5&micro;L MgSO4) and 6 (6&micro;L MgSO4), SRRz is amplified very much. So we decided to use them.
-====PCR Purification====
+=====PCR Purification=====
 {| class="experiments"
 !No.||Name||Concentration||New Name
@@ Line 230: / Line 231: @@
 |6||SRRZ||59.6||SRRz<sub>Sam7</sub>(2)
 |}
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Result
+!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Colony
 |-
 |<partinfo>E0240</partinfo>||1-12-M||1 &micro;L||20||21||LB (Amp+)||rowspan="3"|At 37&#x2103; 7/26 - 7/27||&#xD7;
@@ Line 240: / Line 241: @@
 |<partinfo>J04450</partinfo>||1-5-E||1||20||21||&#xD7;
 |}
-====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
+=====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>=====
-===Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
+====Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi====
-====[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-<partinfo>E0840</partinfo> (Electrophoresis for 35min)====
+=====[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-<partinfo>E0840</partinfo> (Electrophoresis for 35min)=====
 [[Image:KyotoExp100727-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 280: / Line 281: @@
 |}
 Marker: 1kb, 100bp
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 290: / Line 291: @@
 |<partinfo>E0840</partinfo>||120.1
 |}
-====[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]====
+=====[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]=====
 {| class="experiments"
 !Name||Sample||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
@@ Line 300: / 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]]====
+=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Sample||Competent Cells||Total||Plate||Incubation||Result
+!Name||Sample||Competent Cells||Total||Plate||Incubation||Colony
 |-
 |SRRz<sub>Sam7</sub>(1)-B0015|| || || ||rowspan="2"| ||rowspan="2"| ||&#x25CB;
@@ Line 311: / Line 312: @@
-===Wednesday, July 28 <span class="by">By: </span>===
+====Wednesday, July 28 <span class="by">By: </span>====
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 321: / Line 322: @@
 |}
 Diluted S<sub>Sam7</sub>(1)-<partinfo>E0840</partinfo> and S<sub>Sam7</sub>(2)-<partinfo>E0840</partinfo> 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====
+=====[[Team:Kyoto/Protocols#Mutagenesis (Point mutation, Deletion, Insertion)|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 345: / Line 346: @@
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI=====
 {| class="experiments"
 !Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
@@ Line 353: / Line 354: @@
 |S<sub>Sam7</sub>(2)-<partinfo>E0840</partinfo>||3||1||0.1||5.8||10
 |}
-====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min====
+=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min=====
 [[image:KyotoExp100728-1.png|300px|right]]
 {| class="electrophoresis"
@@ Line 370: / Line 371: @@
-===Thursday, July 29 <span class="by">By: </span>===
+====Thursday, July 29 <span class="by">By: </span>====
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
 {| class="experiments"
 !Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
@@ Line 379: / Line 380: @@
 |S<sub>Sam7,&Delta;TMD1</sub>(2)-<partinfo>E0840</partinfo> (1)||50||6||DpnI||0.2||3.8||60
 |}
-====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]====
+=====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]=====
 {| class="experiments"
 !Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
@@ Line 387: / Line 388: @@
 |S<sub>Sam7,&Delta;TMD1</sub>(2)-<partinfo>E0840</partinfo> (1)||2||7||5||1||15
 |}
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Result
+!Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Colony
 |-
 |S<sub>Sam7,&Delta;TMD1</sub>(1)-<partinfo>E0840</partinfo> (1)||3 &micro;L||30||33||rowspan="2"|LB (Amp+)||rowspan="2"|07/29 ~ 07/30||&#x25CB;
@@ Line 397: / Line 398: @@
-===Monday, August 2 <span class="by">By: Wataru, Ken</span>===
+====Monday, August 2 <span class="by">By: Wataru, Ken</span>====
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 412: / Line 413: @@
 |<partinfo>R0011</partinfo>||18.6
 |}
-====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of <partinfo>E0240</partinfo>====
+=====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of <partinfo>E0240</partinfo>=====
 <partinfo>E0240</partinfo> 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 432: / Line 433: @@
 |4&#x2103;||forever||
 |}
-====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
+=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]=====
-====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 441: / Line 442: @@
 |<partinfo>E0240</partinfo>(2)||55.3
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to <partinfo>pSB4K5</partinfo> by 3A assembly====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to <partinfo>pSB4K5</partinfo> by 3A assembly=====
 {| class="experiments"
 !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 449: / Line 450: @@
 |<partinfo>E0240</partinfo>(2) [XP]||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
 |}
-====PCR Purification====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration||Volume
@@ Line 458: / Line 459: @@
 |}
 Stored at -20&#x2103;.
-====Error PCR====
+=====Error PCR=====
 {| class="experiments"
 !Name||Water||MgSO4||dNTPs||10xBuffer||Primer VF2||Primer VR||Template (1)||Template (2)||Template (3)||KOD Pllus ver.2||Total
@@ Line 477: / Line 478: @@
 |4&#x2103;||forever||
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> by DpnI====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> by DpnI=====
-====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+=====[[Team:Kyoto/Protocols#Transformation|Transformation]]=====
 {| class="experiments"
-!Name||Sample||Competent Cells||Total||Plate||Incubation||Result
+!Name||Sample||Competent Cells||Total||Plate||Incubation||Colony
 |-
 |S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (1)||2 &micro;L||20||22||rowspan="3"|-||rowspan="3"|-||&#x25CB;
@@ Line 489: / Line 490: @@
 |}
-===Tuesday, August 3 <span class="by">By: </span>===
+====Tuesday, August 3 <span class="by">By: </span>====
-====Culture====
+=====Culture=====
 Picked two colonies from S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (1), and S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (3), and cultured at 37&#x2103; from 08/03 to 08/04.
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 500: / Line 501: @@
 |<partinfo>R0011</partinfo>||26.8
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
 {| class="experiments"
 !Name||Sample||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 512: / Line 513: @@
 |<partinfo>E0240</partinfo>(2) [XP]||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
 |}
-====PCR Purification====
+=====PCR Purification=====
 {| class="experiments"
 !Name||Concentration
@@ Line 523: / Line 524: @@
 |}
 <partinfo>pSB4K5</partinfo> [EP] is concentrated 10&micro;L and <partinfo>E0240</partinfo>(1) [XP], <partinfo>E0240</partinfo>(2) [XP] are concentrated 1&micro;L.
-====Ethanol Precipitation====
+=====Ethanol Precipitation=====
 After ethanol precipitation, we diluted <partinfo>pSB4K5</partinfo> by 2&micro;L MilliQ
-====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
+=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
 {| class="experiments"
 !Name||Vector||colspan="2"|Insert 1||colspan="2"|Insert 2||Ligation High||Total||Incubation
@@ Line 533: / Line 534: @@
 |<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(2) [<partinfo>pSB4K5</partinfo>]||<partinfo>pSB4K5</partinfo> [EP]||1||<partinfo>R0011</partinfo> [ES]||1||<partinfo>E0240</partinfo>(2) [XP]||1||3||15
 |}
-====[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of <partinfo>I20260</partinfo>====
+=====[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of <partinfo>I20260</partinfo>=====
 {| class="experiments"
 !Name||Water||MgSO4||dNTPs||10xBuffer||Primer VF2||Primer VR||Template <partinfo>I20260</partinfo>||KOD plus ver.2||Total
@@ Line 552: / Line 553: @@
 |4&#x2103;||forever||
 |}
-====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 558: / Line 559: @@
 |<partinfo>I20260</partinfo>||40.6 ng/&micro;L
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
 {| class="experiments"
 !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 564: / Line 565: @@
 |<partinfo>I20260</partinfo> [EP]||45 &micro;L||6||0.6||EcoRI||0.2||PstI||0.2||8||60
 |}
-====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
 {| class="experiments"
 !Name||Concentration||Volume
@@ Line 571: / Line 572: @@
 |}
 <partinfo>I20260</partinfo> [EP] is concentrated at 7&micro;L
-====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
+=====[[Team:Kyoto/Protocols#Ligation|Ligation]]=====
 {| class="experiments"
 !||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
@@ Line 578: / Line 579: @@
 |}
 <div class="measure-construction">
-====Transformation====
+=====Transformation=====
 {| class="experiments"
-!Name||Sample||Competent Cell||Total||Plate||Incubation||Results
+!Name||Sample||Competent Cell||Total||Plate||Incubation||Colony
 |-
 |<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo>(1) [<partinfo>pSB4K5</partinfo>]||1 &micro;L||20||21||rowspan="3"|LB (Kan+)||rowspan="3"|08/03-08/04||&#x25CB;
@@ Line 590: / Line 591: @@
-===Thursday, August 5 <span class="by">By: </span>===
+====Thursday, August 5 <span class="by">By: </span>====
-====Culture and Master Plates====
+=====Culture and Master Plates=====
 <partinfo>pSB4K5</partinfo> 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.
@@ Line 622: / Line 623: @@
 |<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (3)||Green Colony
 |}
-====Sequence====
+=====Sequence=====
 {| class="experiments"
 !Name||Concentration
@@ Line 637: / Line 638: @@
-===Friday, August 6===
+====Friday, August 6====
-====Miniprep====
+=====Miniprep=====
 {| class="experiments"
 !Name
@@ Line 664: / Line 665: @@
 |<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (3)
 |}
-====Restriction Digestion====
+=====Restriction Digestion=====
 {| class="experiments"
 !Name||Sample||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
@@ Line 690: / Line 691: @@
 |<partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>] (3) [EP]||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 |}
-======Electrophoresis=====
+========Electrophoresis======
 {| class="experiments"
 !No.||Name||Length||Results
@@ Line 722: / Line 723: @@
 [[Image:KyotoExp100806-1.png]]
 White colonies are not inserted <partinfo>R0011</partinfo> 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)====
+=====Error PCR (Retry)=====
 {| class="experiments"
 !Name||Water||MgSO4||dNTPs||10xBuffer||Primer VF2||Primer VR||Template S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> failed (50ng/&micro;L)||KOD plus ver.2||Total
@@ Line 743: / Line 744: @@
 |4&#x2103;||forever||
 |}
-====Transformation====
+=====Transformation=====
 {| class="experiments"
-!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Results
+!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Colony
 |-
 |S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (1)||-||4 &micro;L||50||54||rowspan="3"|LB (Kan+)||rowspan="4"|08/06-08/09||&#x25CB;
@@ Line 757: / Line 758: @@
-===Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>===
+====Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>====
-====Miniprep====
+=====Miniprep=====
 {| class="experiments"
 !Name||concentration
@@ Line 766: / Line 767: @@
 |<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo> [<partinfo>pSB4K5</partinfo>]||146.6
 |}
-====Transfotrmation====
+=====Transfotrmation=====
 {| class="experiments"
 !Sample||Sample||colspan="2"|Competent Cell||Total||Plate||Incuvation||Results
@@ Line 774: / Line 775: @@
 |<partinfo>R0011</partinfo>-<partinfo>E0240</partinfo> [<partinfo>pSB4K5</partinfo>]||2||KRX||50||52||&#x25CB;
 |}
-====Restriction Eigestion and Ethanol Precipitation====
+=====Restriction Eigestion and Ethanol Precipitation=====
 To use <partinfo>R0011</partinfo> for next ligation, we digested it by EcoRI and PstI
 {| class="experiments"
@@ Line 782: / Line 783: @@
 |}
 After restriction enzyme digestion, we did ethanol precipitation.
-====Ligation and Transformation====
+=====Ligation and Transformation=====
 {| class="experiments"
-!Name|Sample||colspan="2"|Competent cell||Total||Plate||Incuvation||Results
+!Name|Sample||colspan="2"|Competent cell||Total||Plate||Incuvation||Colony
 |-
 |<partinfo>R0011</partinfo> [<partinfo>pSB4K5</partinfo>, KRX]||2 &micro;L||KRX||50||52||rowspan="2"|LB (Kan+)||rowspan="2"|08/09 20:00-08/10 09:00||&#x25CB;
@@ Line 792: / Line 793: @@
-===Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
+====Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
-====Culture====
+=====Culture=====
 Cultured <partinfo>I20260</partinfo> [<partinfo>pSB4K5</partinfo>, <partinfo>R0011</partinfo>-<partinfo>E0240</partinfo> [<partinfo>pSB4K5</partinfo>], <partinfo>R0011</partinfo> [<partinfo>pSB4K5</partinfo>, KRX], and <partinfo>R0011</partinfo> [<partinfo>pSB4K5</partrinfo>, C2].
-====Minprep====
+=====Minprep=====
 {| class="experiments"
 !Name||Concentration
@@ Line 807: / Line 808: @@
 |S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (2-2)||34.7
 |}
-====Culture and Master Plate====
+=====Culture and Master Plate=====
 At 37&#x2103; 08/09 18:00-08/10 9:00
-===Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>===
+====Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>====
 {| class="experiments"
 !No.||Medium||Cloud||Incubation
@@ Line 844: / Line 845: @@
 |}
 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 <partinfo>R0011</partinfo> [pSB4K5, C2], SRRz 1', 3'====
+=====Miniprep of <partinfo>R0011</partinfo> [pSB4K5, C2], SRRz 1', 3'=====
 {| class="experiments"
 !Name||Concentration
@@ Line 852: / Line 853: @@
 |<partinfo>R0011</partinfo> [pSB4K5, C2] (3)||29.9
 |}
-====Restriction Digestion and electrophoresis of <partinfo>R0011</partinfo> [pSB4K5, C2]====
+=====Restriction Digestion and electrophoresis of <partinfo>R0011</partinfo> [pSB4K5, C2]=====
 {| class="experiments"
 !Name||EcoRI||PstI
@@ Line 886: / Line 887: @@
 [[image:KyotoExp100811-1.png]]
 Each enzyme correctly cut samples.
-====Screening PCR of SRRz====
+=====Screening PCR of SRRz=====
 {| class="electrophoresis"
 !No.||Name||Results
@@ Line 906: / Line 907: @@
-===Thursday, August 12 <span class="by">By: Wataru, Ken</span>===
+====Thursday, August 12 <span class="by">By: Wataru, Ken</span>====
-====Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>====
+=====Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>=====
 {| class="experiments"
 !Name||Template||10xbuffer||100xbuffer||EcoRI||XbaI 1||XbaI 2||SpeI||PstI 1||PstI 2||Water||Total
@@ Line 930: / Line 931: @@
-===Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
+====Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
-====Miniprep of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo>====
+=====Miniprep of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo>=====
 {| class="experiments"
 !Name||Concentration
@@ Line 937: / Line 938: @@
 |S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo>||29.6 ng/&micro;L
 |}
-====Point mutation PCR of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo>====
+=====Point mutation PCR of S<sub>Sam7,&Delta;TMD1</sub>-<partinfo>E0840</partinfo>=====
 {| class="experiments"
 !Name||Template||10xbuffer||dNTPs||MgSO4||Primer Fwd.||Primer Rev.||MilliQ||KOD plus ver.2||Total
@@ Line 958: / Line 959: @@
 |4&#x2103;||forever||
 |}
-====Restriction Digestion by DpnI from 17:50 to 18:50====
+=====Restriction Digestion by DpnI from 17:50 to 18:50=====
-====Electrophoresis====
+=====Electrophoresis=====
 {| class="electrophoresis
 !Name
@@ Line 971: / Line 972: @@
 Marker: Lambda, 100bp
 [[Image:KyotoExp100819-1.png]]
-====Ligation and Transformation====
+=====Ligation and Transformation=====
 {| class="experiments
-!Name||Results
+!Name||Colony
 |-
 |S<sub>&Delta;TMD1</sub>-<partinfo>E0840</partinfo> (1)||&#x25CB;
@@ Line 983: / Line 984: @@
-===Friday, August 20 <span class="by">By: Wataru, Ken</span>===
+====Friday, August 20 <span class="by">By: Wataru, Ken</span>====
-====Making Culture and Master Plate of S<sub>&Delta;TMD1</sub>-<partinfo>E0840</partinfo>====
+=====Making Culture and Master Plate of S<sub>&Delta;TMD1</sub>-<partinfo>E0840</partinfo>=====
-====[[Team:Kyoto/Protocols#Miniprep|Miniprep]====
+=====[[Team:Kyoto/Protocols#Miniprep|Miniprep]=====
 {| class="expeirments"
 !Name||Concentration
@@ Line 991: / Line 992: @@
 |<partinfo>B0015</partinfo>||41.1 ng/&micro;L
 |}
-====[[Team:Kyoto/Protocols#Standard PCR|PCR]] of SRRz====
+=====[[Team:Kyoto/Protocols#Standard PCR|PCR]] of SRRz=====
 {| class="experiments"
 !Name||10xBuffer||MgS04||dNTP||Template||colspan="2"|Primer Fwd.||Primer Rev.||MilliQ||KOD plus ver.2||Total
@@ Line 1,018: / Line 1,019: @@
 |4&#x2103;||forever||
 |}
-====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
+=====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]=====
 {| class="electrophoresis"
 !Name
@@ Line 1,036: / Line 1,037: @@
 [[Image:KyotoExp100820-1.png]]
 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]]====
+=====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]=====
 {| class="experiments"
 !Name||Concentration
@@ Line 1,044: / Line 1,045: @@
 |SRRz (3)||69.0
 |}
-====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
+=====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]=====
 {| class="experiments"
 !Name||Sample||10xBuffer||100xBuffer||EcoRI||XbaI||SpeI||MilliQ||Total||Incubation
@@ Line 1,054: / Line 1,055: @@
 |SRRz (3) [EP]||50||6||0.6||0.4||-||0.4||2.6||60
 |}
-====Purification====
+=====Purification=====
 {| class="experiments"
 !Name||Concentration
@@ Line 1,064: / Line 1,065: @@
 |<partinfo>B0015</partinfo>||25.5
 |}
-====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Transformation]]====
+=====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Transformation]]=====
+----

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 11:52, 23 October 2010

Contents

Notebook

Construction for Lysisbox

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

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

PCR for SRRz and S

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

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

Friday, July 23 By: Wataru, Tomo, Makoto

PCR for SRRz

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

Restriction Digestion and Ligation to insert S gene to <partinfo>E0840</partinfo>

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

PCR Purification

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>

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

Colony PCR of SSam7-<partinfo>E0840</partinfo> (Electrophoresis for 35min)

Wednesday, July 28 By:

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:

Ligation and Phosphorylation

Monday, August 2 By: Wataru, Ken

Standard PCR of <partinfo>E0240</partinfo>

Restriction Digestion for inserting <partinfo>E0240</partinfo> to <partinfo>pSB4K5</partinfo> by 3A assembly

PCR Purification

Error PCR

Restriction Digestion of SSam7,ΔTMD1-<partinfo>E0840</partinfo> by DpnI

Tuesday, August 3 By:

Culture

PCR Purification

Ethanol Precipitation

Standard PCR of <partinfo>I20260</partinfo>

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

Minprep

Culture and Master Plate

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

Miniprep of <partinfo>R0011</partinfo> [pSB4K5, C2], SRRz 1', 3'

Restriction Digestion and electrophoresis of <partinfo>R0011</partinfo> [pSB4K5, C2]

Screening PCR of SRRz

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>

Miniprep of SSam7,ΔTMD1-<partinfo>E0840</partinfo>

Point mutation PCR of SSam7,ΔTMD1-<partinfo>E0840</partinfo>

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

Electrophoresis

Ligation and Transformation

Friday, August 20 By: Wataru, Ken

Making Culture and Master Plate of SΔTMD1-<partinfo>E0840</partinfo>

[[Team:Kyoto/Protocols#Miniprep|Miniprep]

PCR of SRRz

Purification

Ligation and Team:Kyoto/Protocols#Transformation

Colony PCR of S_Sam7-<partinfo>E0840</partinfo> (Electrophoresis for 35min)

Restriction Digestion of S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> by DpnI

Miniprep of S_Sam7,ΔTMD1-<partinfo>E0840</partinfo>

Point mutation PCR of S_Sam7,ΔTMD1-<partinfo>E0840</partinfo>

Making Culture and Master Plate of S_ΔTMD1-<partinfo>E0840</partinfo>

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