PLASMID ISOLATION AND ANALYSIS Part IV DNA Analysis Using Agarose Gel Electrophoresis

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PLASMID ISOLATION AND ANALYSISPart IVDNA
Analysis Using Agarose Gel Electrophoresis
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DNA Analysis Using Agarose Gel ElectrophoresisDay
1
Prepare the agarose gel one day before Part
IV. Review this procedure at the beginning of
the printed protocol or go to the Agarose Gel
Prep in the DNA Fingerprinting protocol on the
ISU biotech web site at www.biotech.iastate.edu
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
1Step 1
Mark four 1.5 ml microcentrifuge tubes as
follows pUC-, pUC, Mini, and Mini-
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
1Step 2
Use the matrix below to mix the reagents. Add
all reagents directly into the solution. Change
tips after each reagent addition.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
1Step 3
Incubate the mixtures in a 37oC water bath for 20
minutes to 1 hour. One hour of incubation yields
the best results.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
1Step 4
Store the reaction in the freezer at -20oC until
you are ready for electrophoresis.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
2Step 1
Thaw the solutions at room temperature and add 4
ul of migration dye in each of the four reaction
tubes.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
2Step 2
Set the micropipettor to 20 ul and draw the
sample from tube pUC-.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
2Step 3
Load the 20 ul of the DNA sample by placing the
pipette tip into the top of the well and slowly
releasing the solution into the well.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
2Step 4
Repeat steps 1 and 2 for the tubes marked pUC,
Mini and Mini-. Add the 1X TBE buffer (70 ml
10X TBE diluted with 630 ml distilled water) to
the electrophoresis chamber making sure to cover
the gel. Run the gels at 150 volts for 1 hour.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
2Step 5
Your instructor will run and stain the gel so you
can view it later.
PLASMID ISOLATION AND ANALYSIS Part IV
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DNA Analysis Using Agarose Gel ElectrophoresisDay
3Results
pUC -
MiniPrep -
pUC -
MiniPrep -
PLASMID ISOLATION AND ANALYSIS Part IV