Biomod/2012/UT/Nanowranglers/Methods: Difference between revisions

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


==DNA Elution following Denaturing or Native PAGE==
==DNA Elution following Denaturing or Native PAGE==

Revision as of 11:05, 22 October 2012

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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 2.5 V of 100% ethanol, 0.1 V of 3 M NaAc (pH 5.2), 2 µL 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 1 mL 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 300 V 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
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