Team:UC Davis/protocols/gelelectrophoresis.html

From 2010.igem.org

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<a name="purpose"><h1>Purpose</h1></a>
<a name="purpose"><h1>Purpose</h1></a>
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Although gel electrophoresis has many purposes, we use them to separate out DNA fragments, specifically the insert from the plasmid. <p>
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Although gel electrophoresis has many purposes, we use them to separate out DNA fragments.  This is done so that we can gel extract wanted DNA parts or simply as a diagnostic to see whether or not we have gotten the right pieces together. <p>
      
      
<a name="references"><h1>References</h1></a>
<a name="references"><h1>References</h1></a>

Latest revision as of 20:37, 10 September 2010

Gel Electrophoresis

Materials

You will need:

  • Agarose
  • 1X TAE
  • Loading Dye
  • 2-log or 100bp Ladder
  • Safestain

Extra Notes

  • Be sure that the well size is large enough (but not too large) to accommodate the amount of DNA you will be loading.
  • Be sure to aware of what concentration your gel is. The difference between a 1% and a 2% agarose gel is significant.

Procedure

 Creating a 1% gel stock
  • Dissolve 5g agarose in 500mL 1X TAE

Creating a 2% gel stock

  • Dissolve 10g agarose in 500mL 1X TAE

When gel is ready to use

  • Have gel trays ready with the gel comb (used to create the wells) in place.
  • Microwave the gel stock. Be sure it is all melted and dissolved.
  • Pour 50mL into one 50mL centrifuge tube. If gel is bigger, pour 100mL into two 50mL centrifuge tubes.
  • When gel solution is lukewarm, pipette 5μL safestain into gel
  • Pour immediately and slowly into gel trays to reduce chunks and bubbles in gel.
  • Wait until gel completely solidifies.

Loading DNA into gel

  • Pipette a 1:5 volume of loading dye to DNA template. (Eg. 1μL loading dye into a 5μL DNA template to be loaded.)
  • Pipette 5μL of ladder into the very first well.
  • Carefully pipette each DNA template to each well.
Running the gel
  • Run electric current between 90V-120V through gel until bands have separated enough.

Purpose

 Although gel electrophoresis has many purposes, we use them to separate out DNA fragments. This is done so that we can gel extract wanted DNA parts or simply as a diagnostic to see whether or not we have gotten the right pieces together.

References
We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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