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From 2010.igem.org

Contents 1 Andreas 1.1 Cloning of N-CPPs into pSB1C3 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.4 ON cultures 1.2 Transfer of m-yCCS into pEX 1.2.1 ON cultures

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

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