Jeff:LabNotes/Microbiome/2011-2-9

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In well MDA using e coli cells with larger well size[edit]

Use new 400 um diameter wells, same procedure, add in REP (Cy3) and ERIC (Cy5) primers. We want to see distinct, nonoverlapping fluorescence. Try and extract amplicons

  • Prior to beginning, create NS and ALS buffers
  • ALS Buffer: 82 uL H20, 10 uL 1M DTT, 8 uL 5 M KOH
  • NS Buffer: 3 mL H2O, 3 mL 2M Tris, 4 mL 1N HCL
  • Make sure an even mix of the 2 buffers has a pH of 7-8 before moving on
  • Clean down hood with ethanol and UV tubes, arrays, gaskets, etc for 10 min
  • Use e coli with initial conc. 10^6 cell/uL, dilute down to 10000 cell/uL
  • Create the following mix: 38uL .5% agarose, 12 uL e coli, 6 uL C12 N6 labeled beads, 4 uL 3% BSA
  • Create another mix with agarose instead of e coli and use as NTC
  • Add 3 uL of mix to square and let settle for 5 min (~6000 cell total)
  • Create a lysis mix: 2uL lysozyme, 2 uL NaCl, 2 uL Tris
  • Add 3 uL of lysis mix to square with cells and let sit for 15 min at rt
  • Add 3 uL of ALS buffer to each and sit for 3 min
  • Create the following master mix while waiting: 15 uL 200 uM N6 primer, 7.5 uL 10x buffer, 17.1 uL H2O, 2.4 uL 25 nM dNTP, 3 uL Phi 29. This master mix is enough for 3 reactions
  • After 3 min, add 3 uL of NS buffer to each square to neutralize
  • Add 16 uL of master mix to each. Incubate at 30 C for 4 hr then 85 C for 3 min, and 4C for 10 min.

Hybridization and Washing

  • Remove secure seal
  • Create following mix: 2 uL 20x SSC, 28 uL Formamide, 10 uL H20 and add 20 uL to each array. UV mix for 10 min. Heat to 70 C. Seal array tightly with new secure seal gasket and incubate at 70 C for 15 min (transfer in pipette tip box). Then 4 C for 10 min
  • UV clean water, Wash 1E, and SSPE for 10 min
  • Wash arrays for 3 min with 20 uL H20 on shaker (transfer and keep in pipette box while shaking)
  • Wash arrays twice with 20 uL Wash 1E for 4 min on shaker
  • Create following mix: 197 uL 6x SSPE with .01% Triton 100 X and 1 uL 16S Cy5 probe, 1 uL ERIC Cy5 probe, 1 uL REP Cy3 probe (all probes have initial conc of 100 uM)
  • Add 20 uL to each square. Cover with new secure seal gasket and incubate at 94 C for 2 min then 54 C for 15 min (use same sterile techniques) and 4 C for 10 min
  • Remove excess solution and wash with 20 uL H2O for 3 min
  • Wash arrays once with 20 uL Wash 1E twice for 4 min on shaker

Results

You can see some overlapping and some non overlapping fluorescence. Again, the Cy5 signal is much stronger. Two amplicons were extracted. I noticed that the beads are hindering extraction. I want to try and dissociate the beads prior to extraction (after washing) with a strong formamide solution. The probes might become unbound from the DNA, but I dont think it matters since they will still be localized in the well. Therefore, any well that has a fluorescent signal should have an amplicon since the excess probes have been washed away.

File:2-9-11 1.jpeg File:2-9-11 2.jpeg Template