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DNA Gel Electrophoresis

DNA Isolation

Restriction Enzyme Digestion

Polymerase Chain Reaction (DNA Polymerase duplicates DNA when cells divide)DNA copying machine creates the compliment strand (ATCG-TAGC)PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a geneRequires certain components:DNA template, contains the region of the DNA fragment to be amplified Two primers, determines beginning and end of the region to be amplifiedDNA-Polymerase, which copies the region to be amplified Nucleotides, from which the DNA-Polymerase builds the new DNA Buffer, chemical environment for the DNA-Polymerase Thermocycler amplification machine

What is PCR?

detection of hereditary diseasesthe id of genetic fingerprintsthe cloning of genespaternity testing. Why Use PCR?

*Note: Each gel chamber holds 25 ml of the Agarose solution.*

Insert the comb and then tape ends of gels with scotch tape. Label the tape with your name.Place gel tray into a clean weigh boat to contain spills.Add 22.5 ml of dH20 and 0.25g of Agarose to a 125 ml Erlenmeyer flask.Microwave until it turns clear and all Agarose is in solution. (roughly 40 seconds-CAUTION: It bubbles quickly so do 10 second intervals)Add 2.5 ml of 10x TAE buffer, then add 20 ml ethidium bromide (EtBr).Gently pour solution into gel tray, remove bubbles and let it sit for 20 minutes.

DNA Gel ElectrophoresisPart I: Making the Gel

Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size, also within the gel What is Gel Electrophoresis and Why Use it?

Our dye (EtBr) is added before

HOW DOES IT WORK?10

-Fill chamber with 2500 ml of 1x TAE-Put gel tray into chamber-Add dye to the extracted/digested DNA -dye weighs the DNA (dye + glycerol)-Load 1st well with 15ml DNA Marker-Load remaining wells with 15ml dye/DNA mixGene Ruler 1kbLoad Your GeldownStability11

Smaller fragments migrate faster

DNAIs (+)

And Theyre Off

3 kb DNA Fragment