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Contents 1 June 1.1 June 21st, 2011 1.2 June 24th, 2011 1.3 June 25th, 2011 1.4 June 28th 1.5 July 8th 1.6 July 10th, 2011 1.7 July 25th, 2011 1.8 July 28th, 2011 1.9 July 29th, 2011 1.10 August 2nd, 2011

June

June 21st, 2011

Restriction, Ligation, and Transformation Lumazine synthase - double terminator and pBAD - RBS into pSB1C3

1. Restriction

• Restricted lumazine synthase - dt out of pSB1K3 and pBAD - RBS out of pSB1T3 with EcoR1 and Pst1
• Restricted destination plasmid pSB1C3 with EcoR1 and Pst1

2. Ligation

• Ligated lumazine synthase-dt into destination plasmid pSB1C3
• Ligated pBAD-RBS into destination plasmid pSB1C3

3. Transformation

• Transformed lumazine synthase - dt in pSB1C3 into DH5∝
• Transformed pBAD-RBS in pSB1C3 into DH5∝

June 24th, 2011

Picked cells from June 21st transformation

June 25th, 2011

Minipreped Cells picked on June 25th, 2011

June 28th

Restriction digest and ran on gel
Lumazine - dt construct successfully inserted into pSB1C3 Glycerol stocked.

Add Gel Picture

July 8th

Performed antibiotic assay on lumazine - dt in pSB1C3
Samples from one colony grew in Kanamicyn; this stock was discarded

PCR

• PCR'd out lumazine - dt
  
• Used Prefix and antisense suffix

Ran samples on Gel to confirm sizes

July 10th, 2011

PCR'd out Lumazine Synthase - Dt and RFP

Ran on a 1% agarose gel for 45min at 70volts

July 25th, 2011

Ran 4 samples on an agarose gel:
D22
T22
D24
T24

Preformed a restriction with EcoRI and PstI for Ryan
Ran on 1% agarose gel

July 28th, 2011

picked cells from plates and ran a Colony PCR of:
k331033-k331035 in pSB1C3
P0440-K331033 in pSB1A2
J04500-LUMAZINE SYNTHASE-DT in pSB1AK3
pBAD-P0440 in pSB1A2
LUMAZINE SYNTASE-DT-pBAD in pSB1A2

July 29th, 2011

Ran PCR samples from July 28th on a 1X TBE Agarose gel

For control comparision of parts from July 28th PCR we ran a PCR of:
K331035
K331033
Lumazine synthase - dt
P0440
Control

EcoRI Restriction of:
J04500-Agn 43 sample 1
J04500 -Agn 43 Sample 2
J04500 - Lumazine synthase - Dt sample 3
K346007
Lumazine synthase - Dt

Ran Restriction on gel to confirm sizes

Notebook team 3 insert picture Page 37

August 2nd, 2011

Transfer of enhanced lumazine synthase from pET-28a into pSB1C3

Restricted with EcoR1 and Pst1

Ran on 1% TAE agarose gel for 90 minutes at 80V