(Difference between revisions)

---

Contents 1 June 1.1 June 21st, 2011 1.2 June 24th, 2011 1.3 June 25th, 2011 1.4 June 28th 2 July 2.1 July 8th 2.2 July 10th, 2011 2.3 July 25th, 2011 2.4 July 28th, 2011 2.5 July 29th, 2011 3 August 3.1 August 2nd, 2011 3.2 August 4th, 2011 3.3 August 5th, 2011 3.4 August 6th, 2011 3.5 August 7th, 2011 3.6 August 8th, 2011 3.7 August 10th, 2011 3.8 August 11th, 2011 3.9 August 16th, 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∝ E. coli cells
• Transformed pBAD-RBS in pSB1C3 into DH5∝ E. coli cells

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

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

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

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 -- unsuccessful.

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 ran at 80V for 90 minutes

Gel Extraction performed

Ligation

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

Transformation

• Transformation of DNA into competent DH5∝ E. coli cells

August 5th, 2011

Transfer of p0440 - K331033 from pSB1A2 into pSB1C3

Restriction

• p0440 - K331033 restricted with EcoR1 and Pst1

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

• Unsuccessful restriction

August 6th, 2011

Transfer of p0440 - K331033 from pSB1A2 into pSB1C3

Restriction

• p0440 - K331033 restricted with EcoR1 and Pst1

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

• Gel melted -- unsuccessful TAE suspected to be the issue

Restriction

• J04500 and P04400-K331033 were restricted with EcoRI and SpeI, Lumazine synthase-Dt-pBAD and K331035 were restricted with EcoR1 and SpeI

Ran Restrictions on 1X TAE agarose Gel

• 8 samples ran total, 2 of each restriction were done, and run in separate lanes, P0440-k331033 showed not bands on Gel

Performed a successful Gel extraction

August 7th, 2011

Ligation

• J04500 and Lumazine- Dt- pBAD in pSB1A2, also ran Controls using Lumazine- Dt- pBAD and 2microL H20

August 8th, 2011

performed a miniprep of August 4th transformation, pBAD+p0440 and Lumazine Synthase-Dt + pBAD

Ran a 1% TBE agarose Gel Results of Gel look good

• attach gel Picture(august 8th pbad p0440 and lumdt pbad psb1c3)

Transformed Enhansed Lumazine Synthase in pSB1C3 and plated

Picked colonies

• J04500 Lumazine synthase Dt in pSB1AK
• Lumazine synthase - Dt- pBAD in pSB1A2
• P0440 - K331033 in pSB1A2 August 9th, 2011

Religated Enhansed Lumazine synthase into pSB1C3

Miniprep'd overnight cultures from august 8th

Transformed enhansed lumazine synthase into Competent Dh5Alpha Ecoli cells

Restricted J04500 - Lumazine-Dt out of pSB1AK3 and P0440 K331033 out of pSB1A2 With EcoR1 and PstI

• Ran 1X TAE Agarose Gel for Restriction
  • Gel ran at 80Volts for 60 minutes
  • Results of gel analysis show peices at wrong sizes, potential restriction issues.

Picked Colonies for overnight culture, from August 8th plating of Enhansed Lumazine synthase

August 10th, 2011

Miniprep

Miniprep'd Enhansed Lumazine from over night culture on August 9th.

Made 1% TAE Agarose Gel set in fridge for use next day.

Ligation

Ligated together

• K331033 - K331035 + pBAD - P0440 in pSB1C3
• I13453- P0440 + Lumazine synthase Construct in pSB1A2

Note: Ligation used 1.0 Micro liters of T4 Ligase in each ligation instead of 0.5 Micro liters

Transformation

Transformed ligation mix into DH5Alpha E.coli Cells

August 11th, 2011

Ran Gel from August 10th, fridge keeps gel well.

Picked Colonies from Gylserol stocks

• P0440 - K331033 in pSB1A2
• pBAD - P0440 in pSB1A2
• Lumazine synthase - Dt - pBAD in pSB1A2
• J04500 - Agn 43 in pSB1AK3

Gel Extraction of J04500 - Lumazine synthase - Dt and P0440 - K331033

Parts Liagated into pSB1C3 and Control Ligation performed with restricted pSB1C3 and ddH20

Ligations were transformed into competent DH5alpha E.coli Cells and Plated

No Colonies grew

August 16th, 2011

Repeated August 11th, Ligations and Transformations of of J04500 - Lumazine synthase - Dt and P0440 - K331033

== Sept 8th, 2011==