IGEM:IMPERIAL/2006/LabCalendar/2006-8-21: Difference between revisions
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*Also the LoxP sites are being PCRed overnight | *Also the LoxP sites are being PCRed overnight | ||
==T9002== | |||
*Left 96-well plate in shaker overnight to see if steady state is reached. | |||
*Readings to be taken tomorrow. | |||
==To do Tomorrow== | ==To do Tomorrow== | ||
Revision as of 12:58, 21 August 2006
- New and Fresh dilution series of AHL: [1000uM, 100uM, 10uM, 5uM, 1uM, 500nM, 100nM, 50nM, 10nM, 1nM]
Maxiprep
- J37031
- J37032
- J37015RS
- 1M B0021
Miniprep
- J37031 & J37032
- Digest with EcoRI & PstI
- 10 μL DNA
- 6 μL ultrapure water
- 2 μL Orange Buffer
- 1 μL each EcoRI & PstI
Ligation
- J37020 test construct = J37031 + J37032
- J37031 (vector) cut with SpeI & PstI, expected size = 999 bp + 3189 bp (pSB1AK3) = 4188 bp
- J37032 (insert) cut with XbaI & PstI, expected size = 938 bp
- J27020 miniprep, expected size = 1945 bp (part length) & 3189 (pSB1AK3)
- 30 μL Digest
- 25 μL DNA
- 3 μL Yellow buffer
- 1 μL of each enzyme
PCR
- Performed on the aiiA predator construct as the correct primers eventually arrived today.
- 10 cycles at 49[[:Category:{{{1}}}|{{{1}}}]]
- 20 cycles at 70[[:Category:{{{1}}}|{{{1}}}]]
- Left to run overnight
- Also the LoxP sites are being PCRed overnight
T9002
- Left 96-well plate in shaker overnight to see if steady state is reached.
- Readings to be taken tomorrow.
To do Tomorrow
- Glass milk purify DNA from gel.
- Ligate insert into vector (J37032->J37031)
- Electroporate into bacteria
- Continue with AiiA ligations (if immunotag fusion PCR is successful).
