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From 2010.igem.org

Contents 1 Mimmi 1.1 MITF-M 1.1.1 Gel 1.2 SOD.his/his.SOD 1.2.1 Ligation 1.3 MITF-M 1.3.1 Colony PCR

Mimmi

MITF-M

Gel

well	sample
1	1kb ladder
2	MITF-M 1
3	MITF-M 2
4	MITF-M 3
5	MITF-M 4
6	blank

• nothing...
  • Make a new colony PCR with DreamTaq

SOD.his/his.SOD

• Weigh the gel slices and add XP2

	tube	gel+tube	gel	XP2	tot
SOD.his	0.99g	1.45g	0.46g	460µl	920µl
his.SOD	0.99g	1.37g	0.38g	380µl	760µl

• Incubate at 60°C for 7m or until the gel has melted

• Follow the E.Z.N.A. gel extraction kit
  • Wash 2 times with SPW wash buffer
  • Incubate 1m and then eluate with 50µl dH₂O

• DNA conc.
  • not able to measure...

Ligation

pSB1C3			pEX		Conditions
Mix	(µl) x2		(µl) x2		time	°C
vector	1		2		10m	22
DNA	14		13
5x buffer	4		4
T4 ligase	1		1
sH2O	0		0
tot	20µl		20µl

MITF-M

Colony PCR

Mix	(µl)	x6		Primers		conditions
sH2O	17.25	103.5		pSB_VF2		time	°C
dNTP	2.5	15		pSB_VR		2m	95
F primer	1	6				30s	95	)
R primer	1	6				30s	55	> 30 cycles
10x buffer	2.5	15				1m20s	72	)
DreamTaq	0.25	1.5				10m	72
DNA	0.5	6x0.5				oo	10
tot	25µl	150µl