Team:Kyoto/Notebook1

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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	Result
<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	Result
<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	Result
<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	Result
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.	S_Sam7(1)-<partinfo>E0840</partinfo>	S_Sam7(2)-<partinfo>E0840</partinfo>	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	Result
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	Result
S_Sam7,ΔTMD1-<partinfo>E0840</partinfo> (1)	2 µL	20	22	rowspan="3"	rowspan="3"	○
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	Results
<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	Results
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	Results
<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	Results
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

====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

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

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