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
September 8th, 2011
Restriction
Restricted parts with respective restiction enzymes
J04500 in pSB1AK3 SpeI and PstI
Enhansed Lumazine synthase-Dt in pSB1C3 XbaI and PstI
J04500-Enhansed Lumazine Synthase in pSB1AK3 EcoRI and SpeI
Dt in psb1AK3 EcoRI and XbaI
Enhansed Lumazine Synthase in pSB1C3 EcoRI and SpeI
Dt in pSB1A2 EcoRI and SpeI
Restriction mixture into freezer overnight after 1 hour at 37 Degrees C
Made a 1X TAE Agarose Gel stored in fridge overnight.
September 9th, 2011
Ran Agarose Gel from September 8th.
Note: more issues with the Gels, Volts too low, Amp too high, Assistance from Weiden lab PhD student, explains in depth how gel rigs, power packs and agarose all work, Helped trouble shoot each area, which lead to Changing the 1X TAE from weiden lab stock. This gave constant Voltage and lowered the amps, Since the agarose gel was made from off TAE Gel had to be run at constant amps for several hours to prevent from melting.
Gel extraction went well all pieces showed up with the exception of J04500-Enhansed Lumazine in pSB1AK3
Set up ligations of
J04500 and Enhansed Lumazine synthase-Dt in pSB1C3
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