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):

  • 104.5uL digest supermix (BSA, NEBuffer 2, water)
  • 1.5uL each of EcoRI and PstI
  • 8uL H2O

(21uL of MM to 4uL DNA.)

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.

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.