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='''Experimental Methods'''=
Making 3% Agarose Gel
# '''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
#: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  
#:Heat container in microwave on medium for 2 minutes  
:Remove, swirl, continue heating on high until all particles are dissolved
#:Remove, swirl, continue heating on high until all particles are dissolved
:Reweigh solution in container, add hot distilled water to restore initial weight, swirl
#: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
#: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
#:Place gel at 4 °C for 20 minutes, then at room temperature for 10 minutes
 
#'''Making/Running 12% Denaturing Gel'''
Making/Running 12% Denaturing Gel
#:'''Making 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
: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
: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
:Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
#:'''Running gel:'''
*Running gel:
#:Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
:Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
#:Denature sample by heating at 90 °C for 5 minutes
:Denature sample by heating at 90 °C for 5 minutes
#:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
#:Clear lanes of excess urea by pipetting  
:Clear lanes of excess urea by pipetting  
#:Pre-run gel at 450 V for 10-20 minutes
:Pre-run gel at 450 V for 10-20 minutes
#:Load samples, run gel at 450 V with a fan
:Load samples, run gel at 450 V with a fan
# '''Making/Running 12% Native Gel'''
 
#:'''Making gel:'''
Making/Running 12% Native 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)
*Making gel:
#:Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
: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)
#:Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
:Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
#:'''Running gel:'''
:Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
#:Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
*Running gel:
#:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
:Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
#:Clear lanes of excess urea by pipetting  
:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
#:Pre-run gel at 300 V for 10-20 minutes
:Clear lanes of excess urea by pipetting  
#:Load samples, run gel at 300 V with a fan
:Pre-run gel at 300 V for 10-20 minutes
#'''Visualizing/Excising Gel'''
:Load samples, run gel at 300 V with a fan
#:'''Visualizing gel:'''
 
#:Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
Visualizing/Excising Gel
#:Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
*Visualizing gel:
#:Scan for Fluorescence Intensity of Storm Imager
:Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
#:'''Excising gel:'''
:Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
#:Print actual size image of gel
:Scan for Fluorescence Intensity of Storm Imager
#:Move gel on top of saran-wrapped PAGE glass plate, place over image
*Excising gel:
#:Use new razor to cut DNA band, place into 1.7 mL tube
:Print actual size image of gel
#'''DNA Elution following Denaturing or Native PAGE'''
:Move gel on top of saran-wrapped PAGE glass plate, place over image
#:'''Following Denaturing PAGE:'''
:Use new razor to cut DNA band, place into 1.7 mL tube
#:Crush isolated DNA with plunger rod until gel is fine
 
#:Suspend gel in 500 µL of 1X TBE
DNA Elution following Denaturing or Native PAGE
#:Place tube on shaking incubator at 80 °C on high for 15 minutes
*Following Denaturing PAGE:
#:Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
:Crush isolated DNA with plunger rod until gel is fine
#:Concentrate DNA by ethanol precipitating flow-through  
:Suspend gel in 500 µL of 1X TBE
#:'''Following Native PAGE:'''
:Place tube on shaking incubator at 80 °C on high for 15 minutes
#:Crush isolated DNA with plunger rod until gel is fine
:Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
#:Suspend gel in 1 mL of 1X TBE
:Concentrate DNA by ethanol precipitating flow-through  
#:Elute DNA overnight at 37 °C
*Following Native PAGE:
#:Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
:Crush isolated DNA with plunger rod until gel is fine
#:Add flow-through to concentrator filter until concentrated down to approximately 50 µL
:Suspend gel in 1 mL of 1X TBE
#'''Ethanol Precipitation'''
:Elute DNA overnight at 37 °C
#: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
:Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
#:Vortex, place in -80 °C freezer for 15 minutes
:Add flow-through to concentrator filter until concentrated down to approximately 50 µL
#: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
Ethanol Precipitation
#:Spin down at 13,000 rpm for 5 minutes, discard supernatant
: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
#:Dry using speed vac for 10-20 minutes
:Vortex, place in -80 °C freezer for 15 minutes
#:Resuspend DNA in sterilized deionized water
:Spin down at 13,000 rpm for 15 minutes at 4 °C
#'''Forming/Checking Hairpins'''
:Remove supernatant from pellet, wash pellet by adding 1 mL of 70% ethanol
#:Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s<sup>-1</sup>
:Spin down at 13,000 rpm for 5 minutes, discard supernatant
#:Incubate strands at 37 °C
:Dry using speed vac for 10-20 minutes
#:Transfer 20 µL of each tube to new tubes
:Resuspend DNA in sterilized deionized water
#:Add 1/5 V of 6X Orange DNA Loading Dye to each tube
 
#:Load 20 µL of each samples into 12% native gel
Forming/Checking Hairpins
#:Run gel at 300 V with a fan
:Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s-1  
#:Visualize DNA
:Incubate strands at 37 °C
#'''Walker System Assembly'''
:Transfer 20 µL of each tube to new tubes
#:'''Substrate 1 (S1) Assembly:'''
:Add 1/5 V of 6X Orange DNA Loading Dye to each tube
#:Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
:Load 20 µL of each samples into 12% native gel
#:Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min<sup>-1</sup>
:Run gel at 300 V with a fan
#:'''Walker (W) Assembly:'''
:Visualize DNA
#: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<sup>-1</sup>
Walker System Assembly
#:'''S1-W Assembly:'''
*Substrate 1 (S1) Assembly:
#:Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
:Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
#:'''Substrate 2 Assembly:'''
:Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
#:Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
*Walker (W) Assembly:
#:Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min<sup>-1</sup>
:Combine  W1-BHQ1, W2-BHQ1
#:'''Final System (S1-W-S2) Assembly:'''
:Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
#:Incubate S2 and S1-W at 37 °C for 3 hours
*S1-W Assembly:
#'''Buffers Used'''
:Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
#:'''10X TBE:'''
:Substrate 2 Assembly
#:Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM) <br>EDTA (F.W. 372.2): 7.44 g (20 mM) <br>Boric acid (F.W. 61.83): 55.03 g (890 mM) <br>Adjust to 1 L with sd H20
:Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
#:Filter with .0.2 μM filter
:Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
#:'''10 X TMgK:'''
*Final System (S1-W-S2) Assembly:
#: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) <br>MgCl2 6H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H2O (1 M) <br>KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H2O (1 M)  
:Incubate S2 and S1-W at 37 °C for 3 hours
#:''Using solutions:''
 
#:Tris (pH 8.0, 1 M): 10 mL (100 mM) <br>MgCl2 (1 M): 4 mL (40 mM) <br>KCl (1 M): 15 mL (150 mM) <br>sd H2O: to 100 mL <br>Filter with .2 uM fliter
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)
<br>
: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

Revision as of 12:22, 22 October 2012

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Experimental Methods

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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)
    Adjust to 1 L with sd H20
    Filter with .0.2 μM filter
    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)
    Using solutions:
    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
    Filter with .2 uM fliter
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