Team:Stockholm/1 October 2010


Revision as of 11:04, 26 October 2010 by Hassanforoughiasl (Talk | contribs)



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.


  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.



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:



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

The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/