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    Extraction of DNA from an Agarose Gel
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    Extraction of DNA from an Agarose Gel

    Introduction

    This is a protocol that you can use for extraction of DNA from an agarose gel - this is important if you need to isolate a particular band - for example, excising and purifying a PCR product that will be used in an assembly reaction, while leaving the original template DNA behind. This protocol is based upon Dr. Camenares's memory - some helpful resources include: https://www.addgene.org/protocols/gel-electrophoresis/, https://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/, https://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/, https://en.wikipedia.org/wiki/TAE_buffer, and http://www.indiana.edu/~lchenlab/protocol_files/agarose_gel_extraction.pdf

    Materials

    • TAE Buffer
      • 40mM Tris, 10mM Acetic Acid, 1mM EDTA
      • 1X Receipe, 1L: 4.844 g Tris, 1.21mL Acetic Acid, 0.372g EDTA
      • 50X Receipe, 1L: 242.2g Tris, 60.5mL Acetic Acid, 18.612g EDTA
    • Agarose Gel, 0.8%
      • 0.8g/100mL of 1X TAE
    • QIAGen Gel Extraction Kit (Buffers QG, PE, etc)
      • A razor blade or equivalent cutting instrument
        • 3M Sodium Acetate (NaAc)
          • 1.5mL microcentrifgue tubes

            Procedure

            • Casting, loading, and running the gel
            1. Prepare or acquire TAE Buffer, melt agaorse in the buffer. Optional: Add Ethidium Bromide to the molten agarose. Optional: Add Guanosine to a 1mM final concentration to the agarose, to protect against UV damage. This is particularily important if the split gel method outlined below is not used.
            1. Pour molten agarose in the mold, allow to cool, and assemble in the tank with buffer.
            1. Load in the following manner (example being given for a 12 well comb):
            • Lane 1 - Ladder, with premixed loading dye
            • Lanes 2 through 5, samples 1 through 4, 5uL DNA + 1uL 6X Loading Dye
            • Lanes 6 and 7 - Skip
            • Lanes 8 through 12, samples 1 through 4, remaining DNA and dye (i.e. 20uL DNA + 4uL 6X Dye)
            • The reason for this loading scheme is to enable the first half of the gel to be used to identify band locations, and the 2nd half for excision and purification.
            1. Run the gel under a moderate voltage to prevent overheating - 80volts for 45 mins.
            • Excision of bands
            1. For each sample or band to be excised, take out a 1.5mL centrifuge tube, measure and record its weight.
            1. Cut the gel in half between lanes 6 and 7. If the entire gel was not already prestained with Ethidium Bromide, stain the 1st half of the gel (the portion with the ladder) for several minutes in an Ethidium Bromide bath.
            1. Visualize the 1st half of the gel (with the ladder) under UV light, and mark the location in each lane of the bands you want excised.
            1. Under normal light, place the two halves of the gel back together side by side, and excise the equivalent band that you targeted in the previous step on the half of the gel that was not exposed to UV. Place each excised gel piece in a 1.5mL centrifuge tube.
            1. Check the accuracy of your excision; visualize the now cut half of the gel under UV light. If any target DNA remains, excise and add to the appropriate tube. Minimize UV exposure in this step!
            1. Weight the tubes that now contain excised gel fragments. Subtract their pre-gel weight to determine the weight of the gel fragment inside. This is necessary for calculations in the QIAgen kit.
            • QIAGen Kit DNA Cleanup.
            1. Follow the instructions from the QIAGen Kit, briefly outlined as follows:
            1. For every 100mg of gel slice, add 300uL of buffer QG to the tube (if your gel slice exceeds 300mg, you will need to split it among two separate tubes or use a larger tube). Incubate for 10 mins at 50C or until gel slice is dissolved. Frequent vortexing may be helpful. Please refer to manual if the color changes orange or violet.
            1. For every 100mg of gel slice, add 100uL of isopropanol and mix
            1. Apply mixture to the QIAGen spin column (successive spins if necessary) Spin and discard flowthrough.
            1. Add 500uL of Buffer QG to the column to wash. Spin and discard flowthrough.
            1. Add 750ul of Buffer PE to the column to wash. Spin and discard flowthrough.
            1. Spin once more, and transfer to a clean 1.5mL centrifuge tube. Elute with the appropriate volume of ddH20 or buffer EB.

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