PLASMID ISOLATION AND ANALYSISPart IV

DNA Analysis Using Agarose Gel Electrophoresis

PLASMID ISOLATION AND ANALYSIS: Part IV

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

DNA Analysis Using Agarose Gel ElectrophoresisDay 1

PLASMID ISOLATION AND ANALYSIS: Part IV

Mark four 1.5 ml microcentrifuge tubes as follows: pUC-, pUC+, Mini+, and Mini-

DNA Analysis Using Agarose Gel ElectrophoresisDay 1Step 1

PLASMID ISOLATION AND ANALYSIS: Part IV

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

Incubate the mixtures in a 37oC water bath for 20 minutes to 1 hour.One hour of incubation yields the best results.

DNA Analysis Using Agarose Gel ElectrophoresisDay 1Step 3

PLASMID ISOLATION AND ANALYSIS: Part IV

Store the reaction in the freezer at -20oC until you are readyfor electrophoresis.

DNA Analysis Using Agarose Gel ElectrophoresisDay 1Step 4

PLASMID ISOLATION AND ANALYSIS: Part IV

Thaw the solutions at room temperature and add 4 ul of migration dye in each of the four reaction tubes.

DNA Analysis Using Agarose Gel ElectrophoresisDay 2Step 1

PLASMID ISOLATION AND ANALYSIS: Part IV

Set the micropipettor to 20 ul and draw the sample from tube pUC-.

DNA Analysis Using Agarose Gel ElectrophoresisDay 2Step 2

PLASMID ISOLATION AND ANALYSIS: Part IV

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.

DNA Analysis Using Agarose Gel ElectrophoresisDay 2Step 3

PLASMID ISOLATION AND ANALYSIS: Part IV

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.

DNA Analysis Using Agarose Gel ElectrophoresisDay 2Step 4

PLASMID ISOLATION AND ANALYSIS: Part IV

Your instructor will run and stain the gel so you can view it later.

DNA Analysis Using Agarose Gel ElectrophoresisDay 2Step 5

PLASMID ISOLATION AND ANALYSIS: Part IV

pUC

- +MiniPrep

+ -pUC

- +MiniPrep

+ -

DNA Analysis Using Agarose Gel ElectrophoresisDay 3Results