Microbiology lab 2

Gel Electrophoresis steps

1) Make the agarose gel 2) Pour into casting tray with comb and allow to solidify 3) Add running buffer, load DNA samples and marker 4) Hook up electric current and run gel at constant voltage until band seperation occurs. Reach bottom. 5) View DNA on UV light box and document results. Stain gel and analyze results

Agarose

A polysaccharide obtained from seaweed (rhodophyceae algal cell walls) that is used as the supporting medium in gel electrophoresis. Hydrogen bonds forms porous matrix

Why does gel electrophoresis separate molecules based on only size and not because of charge (more negative charge, quicker to move)?

All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only.

What buffer was used in gel electrophoresis?

TBE buffer

Why does the DNA migrate towards the anode?

Because the phosphate backbone of DNA is negative and is attracted to the positive electrode.

Buffer is used instead of water when making and running gels because

Buffer enhances the transmission of electric currents in water

AS

CARRIER: Heterozygous, One copy of hemoglobin codes for normal hemoglobin and the other copy codes for sickled hemoglobin. No disease, wont develop it later.

The blue dye migrates from the ___ to the ___

Cathode (-) to the anode (+)

The size of the molecular products is determined by

Comparison with a molecular weight "ladder"

Since stained with dye the DNA fragments can be seen and appear as bands, what does each band represent?

Each band represents a group of same-sized DNA fragments.

Visualizing DNA. How we are able to see DNA.

Ethidium Bromide binds to DNA Fluorescence under UV light makes bands visible

Gel Electrophoresis is used to visualize

Genomic DNA RNA PCR products Plasmids Restriction enzyme digest products

SS

Homozygous for sickled hemoglobin, both copies code for disease, Person suffers from sickle cell anemia. Immune to Malaria.

AA

Homozygous, both copies of hemoglobin code for normal hemoglobin so does not have disease. Not immune to Malaria.

As DNA has a net _______ charge, it migrates towards the gel box's _________.

Negative charge; migrates towards the anode

Is the blue tracking dye negatively or positively charged?

Negatively charged

Gel Electrophoresis is not used to

Separate DNA from the cell or from tissues

Gel electrophoresis separates molecular products based on

Size

When traveling through an agarose gel, larger molecular products will migrate _________ smaller molecular products

Slower than

Why do smaller DNA fragments move faster?

The smaller fragments move faster because they are entangled less in the agarose matrix of the gel

Gel electrophoresis is

a technique used to separate DNA bands (fragments) based on size

Some scientists may want to know which DNA fragments contains a DNA sequence of interest. To do this,

the gel is soaked in a basic solution, so that the double stranded DNA denatures into single strands, then the gel is then transfered to a salt solution and a nylon filter is placed on top of the gel. The salt solution draws the DNA towards the nylon filter, where the DNA adheres to. The filter is then placed in a solution with a radioactive DNA probe. The probe consists of single stranded DNA that is complementary and will hybradize to the band of interest. The filter is then washed to remove and unhybradized probe. X-ray film is placed over the filter while reveals the location of hybridization. Knowing which bands contain the sequence of interest. Band of interest can be cut out of gel.