Using Restriction Enzymes to Confirm the Purified Plasmid DNA is pAMP

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Using Restriction Enzymes to Confirm the Purified Plasmid DNA is pAMP Powered By Docstoc
					                    UsingRestrictionEnzymes Confirm
                     the Purified PlasmidDNA is pAMP

In the bacterialtransformation you showedthat you could createbacteriathat were
resistantto ampicillin by incubatingthe bacteriawith CaClzand the pAMP DNA. You
selected bacteriathat had takenup the pAMP plasmidby plating the bacteriaon LB
agarplatescontainingampicillin. By growing the bacteriafrom one of the ampicillin
resistantcoloniesand then performing a plasmid miniprepprocedureyou were ableto
purify the plasmid DNA from thesebacteria. In this lab you will leam how to perform a
restriction enzpe digestionof a DNA sample. You are using the restrictionenzymes
BamHI and HindIII to confirm that the plasmid you purified is really pAMP. You will
compareyour experimental    resultswith the resultsfrom a computer-simulatedrestriction
enzymedigestto determineif you truly havepurified the pAMP plasmid.


         l.   Get an ice bucket filled with ice and put the tubescontainingthe following
              solutionson ice.
                 ) Your miniprep pAMP DNA

                 ) The 10 X restrictionenzymebuffer neededfor enzymes(seetable).

         2. Get 3 non-sterile1.5ml microfugetubesand label them as follows:

                 Mini - (control)
                 Mini + (both enzymes)
                 BamHI OR HindIII (B or H)
         3. Follow the tablebelow and add the appropriate  solutionsto eachtube.Add the
            restriction enzymeslast. Make surethat all solutionsget addedto the bottom
            of the tube. Changethe pipettetip after every use.

  Tube         Miniprep        l0 X reaction      Bam HI      Ilind III   dHz0      Total
                 DNA             buffer "?"                                        Volume
Mini -            5 rrl             2ul               0          0        13ul     20 ul
Mini +            5pl               2ul              lul        1ul       1 1u l   20 ul
BorH              5pl               2vl                                   l2ul     20ul
       4. Closethe tubesand mix by flicking the tube with your finger. Spin the tubes
          briefly in the picofuge to bring all of the solutionto the bottom of the tube.

       5. Put the tubesin the 37" C water bath, and incubatefor 30-45 minutes.
          Proceed pouring an agarose
                   to                   gel.

Pouring an 0.8 o/oaearosegel

       1. Make a0.8ohagarose

       2. You want to make 50 ml of a0.8o/o
                                          agarose                    will be put
                                                 solution. The agarose
          into 50 ml of lX TAE buffer.

                                     will you need?_
              How many gramsof agarose                               g

       3. Assemblethe gel box in the gel castingposition and insertthe comb. Pour the
          gel and let it harden.

       4. Make 250 ml of 1X TAE buffer solution from the 10X TAE buffer solution.

Preparingthe samplesfor the eel:

       1. When the restrictiondigestis finished,take the tubesout of the water bath and
          give them a brief spin in the picofuge.

       2. Add 4 pl of the 6X DNA loading dye to eachtube, changingpipettetips in
          betweeneachtube.Add 2 pl of SyberGreento eachtube.

       3. Preparethe agarosegel for loading by removing the comb, turning the gel tray
          so that the current will passthrough the gel, and pouring the buffer over the

       4. Load your 72 p.lof eachsampleonto the gel and draw a picture of the gel
          below. Load 8pl marker to first lane.Label the lanesso that you know where
          you loadedeachsample.

       5. Closethe gel box and run the gel at 200 V for 30 minutes.Placepicture of gel
          in your lab book with resultsand conclusion.

       COMPUTER PDRAW PROGRAM: While the gel is running, use the computer
       to print out a simulatedrestriction map of pAMP digestedwith one enzyme and
       also with both enzymes. Tape in your lab book. Also print out a print plot of the
       size of bands and the virtual sel
For your notebook:


    l.   Make the DNA standardcurve as you have done in the past, but only measure
         the distancefrom the bottom of the well to the bottom of eachof the following
         DNA bands in the standard(you will needto refer to the picture of the "Gene
         Ruler I kb DNA ladder" to help you figure out which are the bandsyou want
         to measure).Make a table in your lab notebookwith the sizesof eachof the
         DNA bands and the distance(in mm) that eachband traveled from the well.

            4000 bp
            500 bp

    2 . Plot the distance(in mm) vs the size in bp for eachof the DNA bandslisted

    J.   For the miniprep * samplemeasurethe distancesthat eachof the DNA bands
         traveled and include thesenumbers in your table. Estimate their size in bp
         using the graph.

    4 . Make sure you put the picture of your gel into your lab notebook and label it
         as you have done before (label the laneswith the samplenames,statethe
         percentage agarose
                   of         used,the voltagethe gel was run at and time the gel was


    Include in your conclusionssectionthe map of the pAMP plasmid you createdon
    the computershowing where the BamHI and HindIII sitesare located. Make sure
    you statein the conclusionswhat the estimatedsizesof the DNA bandsfrom the
    mini + samplewere. Compare your results from this experiment to the results that
    were predicted by the computer digest.

    1. Is the plasmid purified truly the pAMP plasmid? How do you know?
    2. Would you have gottenthe samesizeDNA fragmentsif you had cut your
       miniprep DNA with two different restiction enzymes?