Lab Techniques Flashcards
Indirect ELISA
Looks for antibodies in sample using immobilized antigens
Western Blot
Detects target protein of interest using SDS gel electrophoresis and tagged antibody incubation
Sandwich ELISA
Looks for antigen in sample using immobilized antibody w/ captured antigen.
Recombinant DNA
1) Poly T primer used to initiate DNA synthesis from RNA template using reverse transcriptase from viruses
2) Create DNA/RNA hybrid. Degrade RNA w/ RNase H, fill in with DNA polymerase leading to ds cDNA.
3) Digest DNA w/ restriction endonuclease that recognizes palindromic sequences and join w/ DNA ligase to isolate gene of interest.
4) Insert downstream of bacterial promoter
Ex: insulin is post-translationally modified in eukaryotes, but bacteria doesn’t contain eukaryote peptidases to go from preproinsulin –> insulin by removing C chain. Must use two plasmids to make alpha and beta insulin chains separately to form commercial functional insulin, stress under oxidizing conditions so disulfide bridges form between chains and fold correctly.
Southern Blot
Detects changes in DNA by separating based on size – detects insertions or deletions, not point mutations. Run DNA on gel and label with complimentary DNA probe.
Quantitative real time PCR
Measures change in RNA abundance prior to seroconversion.
1) Isolate RNA
2) Synthesize cDNA
3) Measure DNA synthesis by PCR in real time
Northern Blot
Detects size or abundance of RNA by running samples through gel using a probe homologous to region of RNA interested in. Good at detecting alternative splicing (ex: b-thalessemia in which intron is not correctly spliced out, diseases RNA is larger than normal)
SNP Chips
Used to detect multiple SNPs at once. Apply copied and separated DNA to chips with DNA complimentary to gene in question and label nucleotides with different colored fluorescent tags –> add DNA polymerase to continue synthesis of labeled oligonucleotide.
