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Latest revision as of 10:51, 26 October 2010

Contents 1 Mimmi 1.1 flourecent pEX 1.1.1 colony PCR 1.1.2 over expression continue... 1.2 LMWP 1.2.1 glycerol stock + preparation of plasmids

Mimmi

flourecent pEX

colony PCR

Mix	(ml)	X8
sH2O	22.5	180		*Take two samples of respectively clone and add them to 10µl LB
F primer	1	8
R primer	1	8		pEX.BBa_J18930
DNA	0.5	4		pEX.BBa_J18931
tot	25µl			pEX.BBa_J18932

Primers
pEX_VF		conditions			gel
pEX_VR		time	°C		well	sample
		2m	94	1	ladder
		30s	94	)	2	BBa_J18930A
		30s	60	> 30 cycles	3	BBa_J18930B
		2m	72	)	4	BBa_J18931A
		10m	72		5	BBa_J18931B
		oo	10		6	BBa_J18932A
					7	BBa_J18932B
					8	control
					9	ladder

• Well 4 + 7 no product?

Bad mixing?

over expression continue...

• Sonicate the samples on ice. 4X30s with 30s pause in between.

• Heat in 95°C for 1m

• Centrifuge 13000rpm for 1m

• Load 1µl on the comassie mini-gel with a special comb

--> Sample is slimey, DNA in it??

--> Hard to see the differences in the bands

LMWP

glycerol stock + preparation of plasmids

• Start cultures from the PCR colonies

Mix
LB	12ml
Amp	8µl
colony	4µl

• Keep in 30°C ON