"

From 2010.igem.org

Contents 1 Mimmi 1.1 CONTROL 1.1.1 PCR 1.1.2 Gel 1.2 SOD / SOD.his / his.SOD / yCCS 1.2.1 Re-transformation

Mimmi

CONTROL

PCR

pEX.SOD		pEX.yCCS		pEX.SOD.his		pEX.his.SOD
*plasmid		*plasmid 1&2		*plasmid		*plasmid
*Top10 2 clones		*BL21 2 clones from 1&2		*BL21 2 clones		*BL21 2 clones
		*BL21 2 new clones		*Top10 2 clones		*Top10 2 clones

pSB1C3.his.SOD.cTAT

*Top10 6 clones

				conditions
mix	pEX x20	pSB x7		time	°C
mastermix	24.5	24.5		5m	95
DNA	0.5	0.5		30s	95	)
tot	25µl	25µl		30s	55	> 30 cycles
				1m20s	72	)
				5m	72
				oo	10

Gel

well	sample	well	sample
1	ladder	12	ladder
2	pEX.SOD.his	13	pEX.SOD
3	BL21 pEX.SOD.his 1	14	Top10 pEX.SOD 1
4	BL21 pEX.SOD.his 2	15	Top10 pEX.SOD 2
5	Top10 pEX.SOD.his 1	16	pEX.yCCS 5
6	Top10 pEX.SOD.his 2	17	pEX.yCCS 8
7	pEX.his.SOD	18	BL21 pEX.yCCS 1(5) glycerol
8	BL21 pEX.his.SOD 1	19	BL21 pEX.yCCS 2(8) glycerol
9	BL21 pEX.his.SOD 2	20	BL21 pEX.yCCS 1 new
10	Top10 pEX.his.SOD 1	21	BL21 pEX.yCCS 2 new
11	Top10 pEX.his.SOD 2	22	pEX

well	sample
1	ladder
2	pSB1C3.his.SOD.cTAT 1
3	pSB1C3.his.SOD.cTAT 2
4	pSB1C3.his.SOD.cTAT 3
5	pSB1C3.his.SOD.cTAT 4
6	pSB1C3.his.SOD.cTAT 5
7	pSB1C3.his.SOD.cTAT 6
8	pSB1C3.his.SOD

SOD / SOD.his / his.SOD / yCCS

Re-transformation

• Quick transformation
  • 1µl DNA into 500µl BL21 cells
  • Keep on ice
  • Heat shock in 42°C for 50s
  • Cool down on ice
  • Plate on an AMP plate