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アメフラシノート

7/31

Restriction Enzyme Digestion

Nakamura Matsumoto

Sample	Volume	EcoR1	Spe1	Buffer(H)	MilliQ	TOTAL
3/24 PCR(dddD)cloning	3.2	1	1	3	21.8	30
pSB1C3	18.5	1	1	3	6.5	30
pSB1C3(control)	6.2	0	0	0	2.8	10

8/1

Gel Extraction

Yasuda Murata

Lane	DNA
1	1kbp ladder
2	3/24 PCR(dddD)cloning E,S
3	3/24 PCR(dddD)cloning E,S
4	Blank
5	pSB1C3
6	pSB1C3
7	Blank
8	1kbp ladder
9	3/24 PCR(dddD)cloning(control)
10	pSB1C3(control)

8/1

Ligation

Yasuda

Vector	inserter	10xt4LigaseBuffer	t4Ligase	MilliQ	TOTAL
1.5 µL(8/1 pSB1C3 2047bp 13.3µg/mL 20μg)	6.9 µL(8/1 dddD 2545bp 10.8µg/mL 74.60µg)	2µL	1µL	8.6µL	20µL

8/1

transformation

Murata

Name	Sample/(µl)	Competent Cells/(µl)	Total/(µl)	Plate
pSB1C3	3	30	165	SOC(132µl)

8/2

PCR Cloning

Murata

	dddD 121µg/µL
2x KAPA HiFiHotStartReadyMix	12.5µL
F Pre-dddD cloning 62	0.75µL
Rv Spe1-dddDcloning 68	0.75µL
MilliQ	11µL
TOTAL	25µL

8/2

Colony PCR

Honda Matsumoto Nakamura

	Master Mix
Quick Taq	15µL
VF2	0.6µL
VR	0.6µL
MilliQ	13.8µL
TOTAL	30µL

8/2

Restriction Enzyme Digestion

Nakamura Matsumoto

Sample	Volume	EcoR1	Spe1	Buffer	MilliQ	TOTAL
8/2 dddD cloning	8.2	1	1	3	16.8	30
8/2 dddD cloning control	0.41	0	0	1	8.6	10
pSB1C3(control)	6.2	0	0	3(H)	2.8	10

8/3

Electrophoresis for confirmation

Murata

Lane	Sample
1	pSB1C3 dddD 1
2	pSB1C3 dddD 2
3	pSB1C3 dddD 3
4	1kb ladder(3µL)
5	1kb ladder(1µL)

8/3

Gel Extraction

Tatsui

Lane	DNA
1	1kbp ladder
2	Blank
3	pSB1C3 25µμL 2070bp
4	Blank
5	pSB1C3 5µL
6	Blank
7	8/2 cloning dddD 25µL 2544bp
8	Blank
9	3/24 PCR(dddD)cloning(control)
10	8/2 cloning dddD 5µL