Week 10 Lab Review
DNA's Charge
DNA is negatively charged due to the phosphate backbone. This means it will be repelled by negative charge, and attracted by positive charge. NOTE: Since DNA molecules all have the same charge, the migration rate is solely based on their size!
DNA Ladders: Goal
Determine the size of unknown DNA fragments by running gel electrophoresis on them with a corresponding ladder.
Electrophoresis
Is the study of the movement of charged particles in an electric field.
Gel Electrophoresis
It is a technique that separates macromolecules according to their size and charge. The overall charge of a molecule affects the speed at which it travels through the gel Highly charged molecules migrate more quickly through the gel than weakly charged molecules. The mobility of a molecule also depends on its molecular size, shape. The small pores of the gel matrix act as a sieve that provides great resolving power
DNA Ladders: Ladder
Known fragments of DNA are mixed together for use as a standard curve for each gel electrophoresis run.
Characteristics involved in Gel Electrophoresis
Small molecules are able to more easily through the pores than larger molecules and so travel more quickly Large molecules encounter more resistance as they travel through the tiny pores and so travel more slowly Size and net charge are factors that together determine how quickly molecules will travel through the gel and ultimately what their migration distance will be. Small size and strong charge INCREASE a molecules migration rate through the gel Large size and weak charge DECREASE the migration rate
Agarose
a purified form of agar, a gelatinous substance extracted from red algae
Parts of Gel Electrophoresis: Running buffer
keeps gel cool, and conducts electric field through gel
Parts of Gel Electrophoresis: Power Supply
provides constant voltage electric field
Parts of Gel Electrophoresis: Gel rig with electrodes
provides electric field to move DNA
Parts of Gel Electrophoresis: Gel
serves as matrix for DNA to move through