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7: DNA Double Digestion

  • Page ID
    • Nathan Reyna, Ruth Plymale, & Kristen Johnson
    • Ouachita Babtist University & University of New Hampshire

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    DNA Double Digestion Protocol

    Two restriction enzymes are used simultaneously to digest DNA in a single reaction.

    Reagent name Storage Temperature
    Distilled Water Room Temp
    DNA Sample 4°C
    10x NEBuffer 2.1 -20°C
    Restriction Enzyme 1 (EcoRI or XbaI) -20°C
    Restriction Enzyme 2 (SpeI or PstI) -20°C


    • As you add each reagent to a 1.5 ml microfuge tube, stir it in well with the pipet tip and always change your tip in between steps - NEVER double dip your tip!
    • *VERY IMPORTANT!* Do not dispense the liquids onto the sides of the tube. Always dispense the volumes into the very bottom of the tube!
    • Enzymes are stored in glycerol and will never freeze and are ready to be used immediately. They never should be out of the table freezer block or an ice bucket. However, the buffer must be completely thawed before use. (Gently flick the tube and spin the contents to the bottom). You can store the buffers at 4°C but they will only last two weeks. You must keep up with how long your buffer has been at this temperature!

    Procedure: (Take your time! Make sure you’re doing each step correctly!)

    1. Pipette the following into a 1.5 ml microfuge tube: Reagents must be added in the specified order (water is always the first reagent to add!).

    25uL final volume reaction system

    If your DNA concentration is too low you can increase the reaction volume to 40-50 ul.

    X \(\mu L\) of water to get final reaction volume of 25 \(\mu L\)
    X is equal to 25 - (NEB+DNA+Enzyme 1+Enzyme 2)
    2.5 \(\mu L\) NEB buffer 2.1
    \(Y\ \mu L\) DNA
    (usually ~300 ng-Use spectrophotometer to determine concentration and calculate volume needed).
    Y is equal to 300 ng/DNA Concentration(ng/uL)

    \(1\ \mu L\) BioBricks enzyme 1 (either EcoRI or SpeI)
    \(1\ \mu L\) BioBricks enzyme 2 (either XbaI or PstI)

    2. Mix well by pipetting slowly up and down approximately 5x. Be gentle, and do not vortex. Spin the samples for 5 seconds in a balanced microcentrifuge, or flick them to collect the mixture at the bottom of the tube.

    3. Incubate at 37 degrees for at least 1 hour.  Samples can be stored at -20 degrees at this point, but DO NOT forget about step 4 before ligation.

    4. After digestion, incubate your samples for 80°C for 20 minutes (in heat block). For 3A assembly it is important you heat inactivate your samples after digestion. THIS MUST BE DONE or NOTHING ELSE WILL WORK!

    5. Run 5 \(\mu L\) of your digested sample in an agarose gel in order to check that your digestion worked. (See Section 1.4 for making/pouring of agarose gel as well as running a gel electrophoresis).  Make sure you run the proper controls with your samples on the gel: (1) a small volume of uncut DNA for each plasmid digested. (2) Also, always run a lane with DNA Ladder!

    Be sure to clearly label and freeze the remainder of your digestion. This will be used for the ligation step.

    This page titled 7: DNA Double Digestion is shared under a not declared license and was authored, remixed, and/or curated by Nathan Reyna, Ruth Plymale, & Kristen Johnson.

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