Electrophoresis Flashcards
What does electrophoresis allow us to do ?
Separate DNA fragments according to length
What is the electrophoresis separation technique based on ?
The movement of charged molecules in an electric field
What does DNA consist of ?
Two sugar phosphate backbones joined by complementary base pairs
What are phosphates electric charge ?
Negative
Why is DNA attracted to the positive electrode -anode ?
Phosphate backbone is negative
Why are all DNA fragments attracted equally to the anode ?
The charge to mass ratio is uniform throughout its length
What is a gel matrix ?
A cross linked substance that acts as a molecular sieve
What gel matrix is used in DNA electrophoresis ?
A polysaccharide called agarose
What is the purpose of agarose ?
Impedes the migration of DNA molecules
What size molecules find it more difficult to travel through the matrix ?
Larger
What size molecules travel further ?
Smaller
How is the gel matrix made ?
Agarose powder is dissolved in a buffer with a high ion concentration
What is the function of the wells in the matrix ?
Keeps the DNA samples apart
Where is the gel matrix placed ?
Submerged in a high salt buffer
What results in an electrical conductor ?
The high ion concentration in the buffer and the gel itself
What is the purpose of glycerol in loading buffer ?
To make samples dense so they sink to the bottom of wells
What allows us to see what is happening in the matrix ?
Blue stain - Bromo-phenol blue
What type of light is the gel viewed under ?
UV
What does each band of DNA consist of ?
DNA of a particular length
What is the DNA ladder ?
The lane containing the length standards
What is the length of DNA molecules written in terms of ?
The number of base pairs
What is a restriction digest ?
Where DNA is cut into specific fragments
What is gel electrophoresis useful for ?
- Checking restriction digests
- Checking PCR products
- Isolating a desired DNA fragment
What is a restriction enzyme ?
Cuts DNA at a specific base sequence to give a mixture of fragments known as a digest
Is DNA positively of negatively charged ?
Negatively
