Molecular Techniques Flashcards
What are restriction enzymes?
Recognise and Cleave DNA at specific sites - commonly 4, 6, 8 base pair palindromes
What is the function of restriction enzymes in bacteria?
Protect DNA- degrades foreign DNA
What does DNA gel electrophoresis do?
Separate DNA fragments in an electric field based on size
What is a plasmid vector?
A small circular dsDNA found in bacteria
What’s the function of PCR?
To amplify specific DNA fragments
What three things does PCR require?
Thermostable taq polymerase
Primers (in pairs - reverse and forward)
Nucleotides
What three processes happen continually in PCR?
Denaturation
Annealing
Polymerisation
What does protein gel electrophoresis separate proteins on a basis of?
Size
Charge
Shape
What does SDS-PAGE do?
Separates proteins on size. SDS detergent denatures protein (removes secondary and tertiary structure).
What does isoelectric focusing (IEF) do?
Separates proteins based on pI - proteins move through gradient of pH. Migrate unruly pH = pI (no net charge)
What is 2D PAGE?
Separates on charge and size. Separates the bands from IEF into individual proteins
What is proteomics?
Analysis of all proteins in the genome and their functions
What is western blotting?
Following protein gel electrophoresis, bands are transferred onto a membrane (nitrocellulose) and incubated with primary antibody and then labelled secondary antibody
What is southern hybridisation?
Identifying specific DNA sequences using allele specific labelled probes
What is northern hybridisation?
Same as southern but uses RNA on gel
