IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/07/25
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Lock, Key, and Control
7 samples: Lock, Key, Control to be heat inactivated (HI); Lock, Key, Control to be PCR purified (PP); negative control Made up 7x PCR master mix:
Added 20uL master mix to tubes containing 5uL template (0.5ng/uL ultramers), nothing in negative control.
Ran 2nd-strand synthesis cycling protocol:
PCR purified PP samples with Fermentas GeneJet kit, eluting in 25uL. Made a mistake and had to discard the lock.
Heat inactivated HI samples at 80°C for 20 minutes. Later realized that I'm stupid and you can't heat inactivate Taq, but will try and go ahead anyways.
Ran gel of purified samples to assess DNA concentration; as expected PP samples are somewhat lower, but not terribly so.
Made a master mix for HI samples (+ pSB1A3):
(21uL of MM to 4uL DNA.)
For PP samples, individually added:
Incubated at 37°C from 5:00PM - 7:05PM. Heat inactivated at 80°C for 20 minutes.
We ran out of ligase today, so will leave at 4°C until we get some more.