Team:Stockholm/7 September 2010

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 6/9 transformations

Good colony yields on both plates, 1 and 2*.

Colony PCR

4 colonies picked from each plate for verification by colony PCR.

  • 1, 2, 3, 4, 5*, 6*, 7*, 8*
  • PC: pSB1C3.RFP
PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTP, 10 mM 0.4
Fwd primer (VF2) 0.4
Rev primer (VR) 0.4
Cell suspension 0.5
DreamTaq pol. 0.08
  20 μl

Standard colony PCR settings

  • Elongation time: 0:45

Gel verification

Colony PCR gel verification of pSB1C3.N-CPPs
3 μl λ; 6 μl sample.
λ = GeneRuler 50 bp DNA ladder.

1.5 % agarose, 90 V.

Expected bands

  • pSB1C3.Tra10: 389 bp
  • pSB1C3.TAT: 359 bp
  • pSB1C3.LMWP: 368 bp

Results
Relevant bands in clones 3, 4, 6, 7 & 8. Slightly different in size, which could indicate presence of all three N-CPPs. All clones selected for sequencing.

ON cultures

For plasmid prep

  • pSB1C3.N-CPP: pC.NCPP 3, 4, 6*, 7* and 8*
    • 5 ml LB + Cm 25
    • 37 °C, 200 rpm ON

Transfer of m-yCCS into pEX

ON cultures

Set ON cultures of pEX.yCCS for plasmid prep and glycerol stocks. From 3/9 colonies.

  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 37 °C, 200 rpm ON
  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 30 °C