Biomod/2012/UT/Nanowranglers/Methods

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Revision as of 00:10, 22 October 2012 by Chunhua Liu (talk | contribs) (DNA Elution following Denaturing or Native PAGE)
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 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers"><img src="http://openwetware.org/images/c/ce/Concept-dull.png" alt="" /></a>

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Making 3% Agarose Gel

  • In a 250 mL glass container, mix 3 g of Metaphor agarose powder, 100 mL of 1X TBE buffer, then weigh
  • Heat container in microwave on medium for 2 minutes
  • Remove, swirl, continue heating on high until all particles are dissolved
  • Reweigh solution in container, add hot distilled water to restore initial weight, swirl
  • Cool solution to 60 °C, pour into a casting tray, apply a comb
  • Place gel at 4 °C for 20 minutes, then at room temperature for 10 minutes

Making/Running 12% Denaturing Gel

  • Making gel:
    • In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer
    • Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
    • Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
  • Running gel:
    • Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
    • Denature sample by heating at 90 °C for 5 minutes
    • Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
    • Clear lanes of excess urea by pipetting
    • Pre-run gel at 450 V for 10-20 minutes
    • Load samples, run gel at 450 V with a fan

Making/Running 12% Native Gel

  • Making gel:
    • In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd H2O)
    • Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
    • Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
  • Running gel:
    • Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
    • Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
    • Clear lanes of excess urea by pipetting
    • Pre-run gel at 300 V for 10-20 minutes
    • Load samples, run gel at 300 V with a fan

Visualizing/Excising Gel

  • Visualizing gel:
    • Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
    • Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
    • Scan for Fluorescence Intensity of Storm Imager
  • Excising gel:
    • Print actual size image of gel
    • Move gel on top of saran-wrapped PAGE glass plate, place over image
    • Use new razor to cut DNA band, place into 1.7 mL tube

DNA Elution following Denaturing or Native PAGE

  • Following Denaturing PAGE:
    • Crush isolated DNA with plunger rod until gel is fine
    • Suspend gel in 500 µL of 1X TBE
    • Place tube on shaking incubator at 80 °C on high for 15 minutes
    • Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
    • Concentrate DNA by ethanol precipitating flow-through
  • Following Native PAGE:
    • Crush isolated DNA with plunger rod until gel is fine
    • Suspend gel in 1 mL of 1X TBE
    • Elute DNA overnight at 37 °C
    • Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
    • Add flow-through to concentrator filter until concentrated down to approximately 50 µL

Ethanol Precipitation

  • Add 2.5V of 100% ethanol, 0.1V of 3M NaAc (pH 5.2), 2µL of glycogen to initial volume of solution
  • Vortex, place in -80 °C freezer for 15 minutes
  • Spin down at 13,000 rpm for 15 minutes at 4°C
  • Remove supernatant from pellet, wash pellet by adding 1mL of 70% ethanol
  • Spin down at 13,000 rpm for 5 minutes, discard supernatant
  • Dry using speed vac for 10-20 minutes
  • Resuspend DNA in sterilized deionized water

Forming/Checking Hairpins

  • Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s-1
  • Incubate strands at 37 °C
  • Transfer 20 µL of each tube to new tubes
  • Add 1/5 V of 6X Orange DNA Loading Dye to each tube
  • Load 20 µL of each samples into 12% native gel
  • Run gel at 300V with a fan
  • Visualize DNA

Walker System Assembly

  • Substrate 1 (S1) Assembly
    • Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
    • Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
  • Walker (W) Assembly
    • Combine W1-BHQ1, W2-BHQ1
    • Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
  • S1-W Assembly
    • Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
    • Substrate 2 Assembly
    • Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
    • Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
  • Final System (S1-W-S2) Assembly
    • Incubate S2 and S1-W at 37 °C for 3 hours

Buffers Used

  • 10X TBE
    • Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM)
    • EDTA (F.W. 372.2): 7.44 g (20 mM)
    • Boric acid (F.W. 61.83): 55.03 g (890 mM)
    • sd H2O: to 1 L
    • Filtered with .0.2 uM
  • 10 X TMgK
    • Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H2O (1 M)
    • MgCl2 6H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H2O (1 M)
    • KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H2O (1 M)


  • Tris (pH 8.0, 1 M): 10 mL (100 mM)
  • MgCl2 (1 M): 4 mL (40 mM)
  • KCl (1 M): 15 mL (150 mM)
  • sd H2O: to 100 mL
  • Filtered with .2 uM