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

From OpenWetWare
Jump to navigationJump to search
Line 23: Line 23:
==Making/Running 12% Native Gel==
==Making/Running 12% Native Gel==
*Making gel:
*Making gel:
**In a 50mL cnical tube, mix 15mL of 40% Acyrlamide, 5mL of 100% Glycerol, 5mL of autoclaved 10X TBE, 25mL of sterilized deionized water (sd H2O).
**In a 50mL cnical tube, mix 15mL of 40% Acyrlamide, 5mL of 100% Glycerol, 5mL of autoclaved 10X TBE, 25mL of sterilized deionized water (sd H2O)
**Add 500µL of 10% APS and 50µL of TEMED, mix thoroughly by inverting.
**Add 500µL of 10% APS and 50µL of TEMED, mix thoroughly by inverting
**Immediately pour gel solution into glass plate assembly, apply comb, wait for gel to solidify.
**Immediately pour gel solution into glass plate assembly, apply comb, wait for gel to solidify
*Running gel:
*Running gel:
**Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye.
**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.
**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 300V for 10-20 minutes.
**Pre-run gel at 300V for 10-20 minutes
**Load samples, run gel at 300V with a fan.
**Load samples, run gel at 300V with a fan


==Visualizing/Excising Gel==
==Visualizing/Excising Gel==

Revision as of 23:46, 21 October 2012

<html> <style type="text/css"> //<!--

//Get the playbill font by Hank Gillette, 1992, public domain @font-face { 

 font-family: Playbill2;  
 src: url( 'http://openwetware.org/images/e/e6/PLAYBILL.eot' ); /* IE */  
 src: local(Playbill), 
      url( 'http://openwetware.org/images/a/a1/PLAYBILL.TTF' ) format('truetype'),
      url( 'http://openwetware.org/images/b/ba/PLAYBILL.svg#font' ) format('svg'); /* non-IE */

} /* Define the background of the whole page */ @media screen { 

   body { background: #c5d0ea 0 0 no-repeat; }

} /* Define the div holding the page contents */

  1. globalWrapper{
 left: 50%;
 height: 100%;
 background-color: #FFFFFF;
 width: 900px;
 margin-left: -450px; /* Should be width / 2 */
 margin-top: 0px;
 margin-bottom: 0px;
 margin-right: 0px;
 padding: 0 0 0 0;   
 position: absolute;

} .toc{ 

 display:none;

} /* Hide the page heading at top */ .firstHeading {display:none;} /* Hide the bar at the left */

  1. column-one {display:none;}

/* Define the div holding the page contents (this div is inside globalWrapper. We override its default margins) */

  1. content{
 margin: 0 0 0 0;
 font: normal 100% sans-serif;
 color: #000033;
 padding: 12px 12px 12px 12px;
 background-image: url(http://openwetware.org/images/3/38/FortDavis-Pano-Cropped.jpg); background-repeat: no-repeat; 
 border: 0;

} /* Miscellaneous stuff. Feel free to add style definitions. */ h3 { 

 /*
 color: #CC5500;
 font: normal 3em sans-serif;
 letter-spacing: 1px; 
 margin-bottom: 0; 
 */
 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

} h2 { 

 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

} h1 { 

 /*
 color: #CC5500;
 font: normal 3em sans-serif;
 letter-spacing: 1px; 
 margin-bottom: 0; 
 */
 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

}

.scriptFont { 

  color: #0;
  padding-top: 5px;
  font: normal 1em Georgia;

}

/* Code taken from publicly available css tutorials at onderhond.com */ .linkconcept a {background:url("http://openwetware.org/images/0/03/Concept-bright.png") left top no-repeat;} .linkmembers a {background:url("http://openwetware.org/images/2/24/Members-bright.png") left top no-repeat;} .linkresults a {background:url("http://openwetware.org/images/a/ac/Results-bright.png") left top no-repeat;} .linkmethods a {background:url("http://openwetware.org/images/0/02/Methods-bright.png") left top no-repeat;} .linkrefs a {background:url("http://openwetware.org/images/2/28/References-bright.png") left top no-repeat;}

.linkgeneral {float:left; padding-right:3px; } .linkgeneral a {display:block; width:141px; height:52px;} .linkgeneral a:hover img {position:absolute; left:-999em; top:-999em;} .linkgeneral a:hover {zoom:1;} //--> </style> <div style="text-align:center" /> <img src="http://openwetware.org/images/5/59/NanoWranglers.png"/><br /> <div class="scriptFont"> Undergraduate DNA nanotechnology research group from the University of Texas at Austin </div> <img src="http://openwetware.org/images/e/e6/NanoWranglersDNAStripe.png"/><br /> <!-- links at top --> <div style="float:left; width:85px; height:52px; display:block" > </div> <div class="linkconcept linkgeneral"> 

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers"><img src="http://openwetware.org/images/c/ce/Concept-dull.png" alt="" /></a>

</div> <div class="linkmethods linkgeneral"> 

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Methods"><img src="http://openwetware.org/images/b/be/Methods-dull.png" alt="" /></a>

</div> <div class="linkresults linkgeneral"> 

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Results"><img src="http://openwetware.org/images/8/8f/Results-dull.png" alt="" /></a>

</div> <div class="linkmembers linkgeneral"> 

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Members"><img src="http://openwetware.org/images/4/47/Members-dull.png" alt="" /></a>

</div> <div class="linkrefs linkgeneral"> 

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/References"><img src="http://openwetware.org/images/4/49/References-dull.png" alt="" /></a>

</div> <div style="clear: both;" ></div> <img src="http://openwetware.org/images/e/e6/NanoWranglersDNAStripe.png"/><br />

</div> </html>

Making 3% Agarose Gel

  • In a 250 mL glass container, mix 3g 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, a apply 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 50mL conical tube, mix 30mL of 20% Acrylamide in 1X TBE, 20mL of 1X TBE Denaturing PAGE dilution buffer.
    • Add 500µL of 10% APS and 50µL 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 450V for 10-20 minutes.
    • Load samples, run gel at 450V with a fan.

Making/Running 12% Native Gel

  • Making gel:
    • In a 50mL cnical tube, mix 15mL of 40% Acyrlamide, 5mL of 100% Glycerol, 5mL of autoclaved 10X TBE, 25mL of sterilized deionized water (sd H2O)
    • Add 500µL of 10% APS and 50µL of TEMED, mix thoroughly by inverting
    • Immediately pour gel solution into glass plate assembly, apply 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 300V for 10-20 minutes
    • Load samples, run gel at 300V with a fan

Visualizing/Excising Gel

  • Visualizing gel:
    • Add 5µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50mL 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 1.7mL tube

DNA Elution following Denaturing or Native PAGE

  • Following Denaturing PAGE:
    • Crush isolated DNA with plunger rod until gel is fine
    • Suspend gel in 500µL 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 1mL 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 50µL

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 .0.2uM
  • 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 (1M)
    • MgCl26H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with dd H2O (1M)
    • KCl (F.W.74.55): 7.455 g, adjust to 100 mL with dd H2O (1M)


  • Tris (pH 8.0, 1M): 10 mL (100 mM)
  • MgCl2 (1M): 4 mL (40 mM)
  • KCl (1M): 15 mL (150 mM)
  • sd H2O: to 100 mL
  • Filtered with .2uM
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2012/UT/Nanowranglers/Methods&oldid=638261"