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From 2010.igem.org

Contents 1 Andreas 1.1 Gel verification 1.2 ON cultures 2 Mimmi 2.1 SOD / SOD.his / his.SOD / yCCS 2.1.1 Gel

Andreas

Gel verification

Of 21/9 colony PCR samples

1 % agarose, 120 V

Expected bands

• pSB1A2.RBS⋅yCCS (pA.RBSy): 1018 bp
• pEX.N-TAT⋅SOD⋅His (pEX.nTSH): 735 bp
• pSB1C3.N-LMWP⋅SOD⋅His (pC.nLSH): 857 bp
• pSB1K3.N-LMWP⋅SOD⋅His (pK.nLSH): 857 bp
• pSB1C3.SOD⋅His (PC): 815 bp

Results

• pSB1A2.RBS⋅yCCS: Seemingly correct-sized bands for clones 2, 3 and 4.
• pEX.N-TAT⋅SOD⋅His: Seemingly correct-sized bands for clones 3 & 4.
• pSB1C3.N-LMWP⋅SOD⋅His: Seemingly correct-sized bands for clones 1 & 4.
• pSB1K3.N-LMWP⋅SOD⋅His: Seemingly correct-sized band for clone 1.

ON cultures

1. 5 ml LB + appr. antibiotic (100 Amp, 25 Cm or 50 Km); 37 °C, 225 rpm
   • pSB1A2.RBS⋅yCCS: clones 3 and 4
   • pEX.N-TAT⋅SOD⋅His: clones 3 and 4
   • pSB1C3.N-LMWP⋅SOD⋅His: clones 1 and 4
   • pSB1K3.N-LMWP⋅SOD⋅His: clone 1

***Note to self***

It might be possible that pSB1A2.RBS⋅yCCS clones have been mixed up with pEX.N-TAT⋅SOD⋅His. After plasmid prep, this should be tested by digesting the samples and analyze digestion sizes.

Mimmi

SOD / SOD.his / his.SOD / yCCS

colony PCR				samples		Conditions
				pEX.SOD x3		time	°C
mix	(µl)	x13		pEX.SOD.his x3		5m	95
mastermix	24.5			pEX.his.SOD x3		30s	95	)
DNA	0.5			pEX.yCCS x3		30s	55	> 30 cycles
tot	25µl					1m20s	72	)
						5m	72
						oo	25

Gel

well	sample	well	sample
1	ladder	9	ladder
2	pEX.SOD.his 1	10	pEX.SOD 1
3	pEX.SOD.his 2	11	pEX.SOD 2
4	pEX.SOD.his 3	12	pEX.SOD 3
5	pEX.his.SOD 1	13	pEX.yCCS 1
6	pEX.his.SOD 1	14	pEX.yCCS 2
7	pEX.his.SOD 1	15	pEX.yCCS 2
8	pEX.SOD.his	16	-