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From 2010.igem.org

Contents 1 Andreas 1.1 LB agar plates 1.2 Assembly of His⋅SOD⋅cCPP constructs 1.2.1 Digestion 2 Nina 2.1 Miniprep 2.2 Send for sequencing 2.3 Overnight culture 2.4 iGEM Uppsala collaboration 3 Mimmi 3.1 Over expression

Andreas

LB agar plates

• 20 x 100 μg/ml Amp LB agar plates

Assembly of His⋅SOD⋅cCPP constructs

Digested pMA.His⋅SOD constructs for later assembly into cCPP plasmids, digested by Johan.

Digestion

	pMA.His⋅ SOD
10X FastDigest buffer	3
DNA (3 μg)	7.5
dH2O	17.5
FD EcoRI	1
FD AgeI	1
	30 μl

• Incubation: 37 °C, 0:30
• Inactivation: 80 °C, 20 min

Stored in -20 °C for later ligation.

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Nina

Miniprep

I performed a miniprep on IgG protease_Tra10_Ntermin#4 and Fusion_NS#2 according to the procedure that is described in protocols.

Send for sequencing

I sent two samples for seqeuncing. The mixture was 15 ul sample and 1.5 ul forward bank vector verification primer VF.

• IgG protease_Tra10_Ntermin#4 ASB0045 682

• Fusion_NS#2 ASB0045 681

Overnight culture

I inoculated 12 ml LB and 24 ul chloramphenicol with IgG protease_Tra10_Ntermin#6 again since I acidentally droped the previous sample and therefore lost it.

iGEM Uppsala collaboration

I drove to Uppsala and started a collaboration with their team. I obtained a construct that they want me/our group to PCR with verification primers (VF2 & VR) and run on an agarose gel. I in turn gave them the tyrosinase gene to also amplify via PCR but with own designed primers.

• Uppsala iGEM team's PCR master mix and prgm:

Mimmi

Over expression

• Start culture (9:30)
  • 20ml LB + 200µl culture from ON

• At OD=0.6 add IPTG 1mM (checked at 12:00, allready too high OD, had to dilute...)

• Take samples at
  • 0h
  • 30min (50min)
  • 3h

• Spinn down and resuspend in 100µl SDS-buffer
  • dilute 3h samples 1:4
  • heat at 95°C for 10min
  • freeze

• Save pellet from the culture to purify protein