Team:UI-Indonesia/August
From 2013.igem.org
August
August 5th
- Temperatures used for the PCR are 510C, 54.50C, 60.20C, 63.90C, 670C and 69.50C
- The bands were very thin, so we decided to re-PCR the alpha and omega fragment
August 6th
- Temperatures used for the PCR are 500C, 53.20C, 55.50C, 61.00C, 66.80C, and 68.40C
- The thickest band was shown at 53.20C for alpha fragment and at 500C for omega fragment. Hence the temperatures said above will be used for PCR using Taq HiFi
August 12th
- We got bands with correct length, however we got thin bands on well containing omega fragment of beta galactosidase. We decided to do large scale PCR using PFx the next day.
August 13th
- Running electrophoresis
- We got the correct length for all fragments
August 14th
- Running electrophoresis:
- Correct length for all fragments
- Restricting pQE80L using PstI
- We use three tubes, each of them containing PstI with different volume: 0.5 μL, 5 μL, and without any enzyme added for negative control.
- Running the restricted PCR
- The result shows that the PstI is no longer in a good condition. For 1 hr incubation period, the restriction using 0.5μL PstI does not produce the correct length of pQE 80L (4751 bp)
- Incubating the restricted pQE80L overnight
- This step was done in order to check the activity of the enzyme in extended period.
August 15th
August 16th
August 17th
- Alpha fragment concentration: 17.1 ng/μL
- Omega fragment concentration: 18.8 ng/μL
- Omega+linker fragment concentration: 17.1 ng/μL
August 18th
August 19th
August 20th
August 22nd
August 23rd
August 24th
August 25th
- LMA omega fragmetns
- Lox511 gBlock
- (G4S)4 gBlock
- Omega fragment-pCDNA 3.1
- Alpha fragment-pCDNA 3.1
- Omega fragment-pSB1C3
- Lox511-pSB1C3
- (G4S)4- pSB1C3
- Omega fragment restricted with EcoRI and PstI
- Lox511
- (G4S)4
August 26th
- Lox511
- (G4S)4
- pQE 80-L
- pQE 82-L
- Positive control for ampicillin and chloramphenicol antibiotics