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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of operon constructs into pEX 1.1.1 Plasmid prep 1.1.2 Digestions 1.1.3 Ligations 1.1.4 Transformation 1.2 nCPP culture growth assay 2 Mimmi 2.1 clone non-CPP operon 2.1.1 Transformation

Andreas

Transfer of operon constructs into pEX

Continued Mimmi's experiments'

Plasmid prep

• 40 μl elution buffer (x2)

DNA concentration
Sample	Conc [ng/μl]	A260/A280
pSB1C3.SOD.yCCS	188.2	1.94
pSB1C3.SOD⋅His.yCCS	223.6	1.91
pSB1C3.His⋅SOD.yCCS	230.6	1.93

Digestions

	pSB1C3. SOD.yCCS	pSB1C3. SOD⋅His.yCCS	pSB1C3. His⋅SOD.yCCS
10X FastDigest buffer	2	2	2
DNA (1 μg)	5.3	4.5	4.3
dH2O	10.7	11.5	11.7
FD XbaI	1	1	1
FD PstI	1	1	1
	20 μl	20 μl	20 μl

• Incubation: 37 °C, 30 min
• Inactivation: 80 °C, 20 min

Ligations

Vector: pre-digested pEX.RFP (X+P)

	pEX.SOD. yCCS	pEX. SOD⋅His. yCCS	pEX. His⋅SOD. yCCS
10X T4 Ligase buffer	2	2	2
Vector DNA	3	3	3
Insert DNA	9	9	9
dH2O	5	5	5
T4 DNA ligase	1	1	1
	20 μl	20 μl	20 μl

• Incubation: 22 °C, 15 min

Transformation

By Mimmi

nCPP culture growth assay

Prepared new clones to repeat the experiment from 15/10 by transforming new BL21.

Quick transformation protocol.

• 50 μl competent BL21 (30 μl plated)
• 0.5 μl plasmid DNA
  • pEX.SOD⋅His
  • pEX.nTra10⋅SOD⋅His
  • pEX.nTAT⋅SOD⋅His
  • pEX.nLMWP⋅SOD⋅His

Mimmi

clone non-CPP operon

(Andreas digested the operon from the vector pSB1C3 and ligated into pEX)

Transformation

• 66µl thawed cells + 2µl ligated vector

• Incubate on ice 30min
• Heat shock 55s in 42°C, cool down on ice
• Add 900µl LB, incubate in 37°C for 1h
• Spinn down cells and remove 900µl supernatant
• Resuspend cells and plate on LB_amp + 0.1M IPTG (50µl)