Download DNA-Polymerase

•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 gene
•Requires 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 amplified
DNA-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
•detection of hereditary diseases
•the id of genetic fingerprints
•the cloning of genes
•paternity testing.
*Note: Each gel chamber holds 25 ml of the Agarose solution.*
1. Insert the comb and then tape ends of gels with scotch
tape. Label the tape with your name.
2. Place gel tray into a clean weigh boat to contain spills.
3. Add 22.5 ml of dH20 and 0.25g of Agarose to a 125 ml
Erlenmeyer flask.
4. Microwave until it turns clear and all Agarose is in
solution. (roughly 40 seconds-CAUTION: It bubbles
quickly so do 10 second intervals)
5. Add 2.5 ml of 10x TAE buffer, then add 20 ml ethidium
bromide (EtBr).
6. Gently pour solution into gel tray, remove bubbles and let
it sit for 20 minutes.
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
Our “dye”
(EtBr) is
added
before
-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 mix
Gene Ruler 1kb
Smaller
fragments
migrate faster
DNA
Is
(+)
3 kb DNA
Fragment