IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/07/25

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Lock, Key, and Control

 * Since we seem to be having contamination problems with the assembled lock/key/control in pSB1A3, tried it again from the start.

Second-strand Synthesis
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:
 * 3.25uL G1004 primer (10uM)
 * 3.25uL G1005 primer (10uM)
 * 16.25uL 10X ThermoPol buffer
 * 0.65uL Taq
 * 16.25uL 2mM dNTPs
 * 90.35uL H2O

Added 20uL master mix to tubes containing 5uL template (0.5ng/uL ultramers), nothing in negative control.

Ran 2nd-strand synthesis cycling protocol:
 * 94°C 2 minutes
 * (Repeat steps between brackets 25x)
 * [[94°C 15 seconds
 * 58°C 15 seconds
 * 72°C 15 seconds
 * 72°C 2 minutes]]
 * 4°C forever

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.

Restriction Digest
Made a master mix for HI samples (+ pSB1A3): (21uL of MM to 4uL DNA.)
 * 104.5uL digest supermix (BSA, NEBuffer 2, water)
 * 1.5uL each of EcoRI and PstI
 * 8uL H2O

For PP samples, individually added:
 * 0.5uL each of EcoRI and PstI
 * 2.5uL 10X BSA
 * 2.5uL NEBuffer 3
 * 19uL DNA.

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.
 * Amelia Hardjasa 22:51, 25 July 2009 (EDT)

Jammer

 * Looking back at gels, I am not sure we overnighted/miniprepped/glycerol stocked the correct jammer.
 * Redoing a colony PCR with 7 colonies (including 3 of the old ones I cPCRed) from the 090717 transformation plate.
 * Recipe: 85uL PCR supermix + 0.5uL Taq, 10uL in each tube.
 * Touch tip to transformant, touch to spot plate, then swirl in tube.
 * Standard 1kb VF2/VR cycling protocol.