Team:Stockholm/16 October 2010

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Contents

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 LBamp + 0.1M IPTG (50µl)





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