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

• Gel melted

colonies picked see lab book team 3/ team 1 pg.42

August 4th, 2011

Restriction

• Enhanced lumazine synthase restricted with EcoR1 and Pst1
• pSB1C3 restricted with EcoR1 and Pst1
• pBAD - P0440 restricted with EcoR1 and Pst1
• lumazine - dt-pBAD -RBS restricted with EcoR1 and Pst1

Gel from August 2nd re-done

• Ran at 80V for 90 minutes

Gel Extraction performed

Ligation

• Enhanced lumazine with pSB1C3
• pBAD - p0440 with pSB1C3
• lumazine - dt -pBAD - RBS with pSB1C3