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From 2010.igem.org

Contents 1 Andreas 1.1 Plasmid preps 2 Mimmi 2.1 SOD / yCCS 2.1.1 over expression 2.1.2 PhastGel 2.2 his.SOD.cTAT 2.2.1 colony PCR 3 Johan

Andreas

Plasmid preps

From 16/9 ON cultures

DNA concentration
Sample	Conc [ng/μl]	A260/A280
pSB1C3.N-TAT	64.07	n/a
pSB1C3.N-Tra10	46.38	n/a
pSB1C3.N-LMWP	65.77	n/a
pSB1K3.N-TAT⋅SOD⋅His 4	87.83	n/a
pSB1K3.N-TAT⋅SOD⋅His 5	8.464	n/a
pSB1K3.N-Tra10⋅SOD⋅His 5	6.872	n/a
pEX.SOD	31.57	n/a

Mimmi

SOD / yCCS

over expression

• Start culture
  • 10ml LB_AMP + 100µl old culture (8:30)

• Measure OD=0.6 (11:30)
  • At OD=0.6 add IPTG 1mM

• Take samples after:
  • 0h
  • 2h
    • take 500µl, spinn down and remove LB
    • resuspend in 50µl SDS buffer
    • Heat in 95°C, 5min
    • Freeze
    • Re-heat in 95°C, 5min

• Run gel

PhastGel

well	sample			well	sample
1	ladder			1	ladder
2	SOD.his 0h			2	yCCS 1 0h
3	SOD.his 2h			3	yCCS 1 2h
4	his.SOD 0h			4	yCCS 2 0h
5	his.SOD 2h			5	yCCS 2 2h
6	ladder			6	ladder

his.SOD.cTAT

colony PCR

mix	(µl)	x8		Primers		conditions
mastermix	24.5			pSB1_VF2		time	°C
DNA	0.5			pSB1_VR		5m	95
tot	25µl					30s	95	)
						30s	55	> 30 cycles
						1m20s	72	)
						10m	72
						oo	25

Johan

PCR again, to see if the 900 nt-band or the 700 nt-band is correct

0,5 µl pol

0,5 µl dNTP

5 µl 5x buffer

1,5 µl for primer

1,5 µl rev primer

16 µl H2O