IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-12

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Results from 7/11 Thrombin-Binding Experiment

  • gels removed at 10:00 am: appear to have run off the gel (very hard to tell since they were so faint to begin with)
  • removed gels from plastic
    • cut bottom of gel where it sticks out of narrow hole at the bottom
    • pried two plastic sides apart (gel now stuck to one side)
    • loosen top edge by sliding flat tool under it a bit.
    • make sure the very bottom is completely cut
    • run it under water over the a plastic tupperware container - move it back and forth left to right - eventually the gel should come free into the container.
    • hold the gel in the container while you pour the water out.
  • stained with GelCode Blue Stain (Coomassie blue) (in brown bottle in first 4[[:Category:{{{1}}}|{{{1}}}]] fridge)
    • pour enough Coomassie blue over it to cover the gel.
    • put the lid on the tupperware and put it on the rocker for about 20 minutes.

Protein- and DNA-staining control experiment

  • goal: to load varying concentrations of thrombin onto a gel and stain with Coomassie blue in an attempt to image the protein, and various concentrations of DNA onto a gel and stain with a yet-to-be determined stain to image the DNA
Tube Lane Thrombin mass (μg) 2 μM thrombin (μL)
(2 μM = 1.299 μg/μL)
water (μL) 10x loading dye (μL) total volume (μL)
110.650.58.52.011.0
221.31.08.02.011.0
332.62.07.02.011.0
443.93.06.02.011.0
555.24.05.02.011.0
Tube Lane 2 μM Aptamer (μL) water (μL) 10x loading dye (μL) total volume (μL)
171.08.03.012.0
282.07.03.012.0
393.06.03.012.0
4104.05.03.012.0
5115.04.03.012.0
  • all reagents were mixed in individual 0.2 mL PCR tubes
  • 10-20% Invitrogen polyacrylamide gel run at 25 V for 2.5 h.
Why today was a complete bust (photo shows run/stop in correct configuration)
Why today was a complete bust (photo shows run/stop in correct configuration)
  • gel catridge opened and cut between protein and DNA sections
    • was cut before removal from one side of cartridge and led to some tearing during removal from that side, cut after removal next time
  • protein section was rocked in GelCode Blue Stain for 1 hr.
  • DNA section was rocked in 100 mL Tris-glycine buffer with 10 μL of 10 mg/mL ethidium bromide for 1 hr.
  • results
    • loading dye diffused about 3 mm in all directions into the gel
    • neither the protein nor the DNA imaged on the gel
  • discussion
    • when you don't press the "run/stop" button on the power supply so that the light next to "run" is lit, you're not supplying any power for electrophoresis. we hadn't.
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