Methods Flashcards
Sanger advantages
Cheap, fast, up to 1000bp reads
Sanger disadvantages
You need to have a known seq for primer, only one seq at a time
What can mess up your sanger results?
polymerase fidelity, multiple primer binding sites, allelic variation
how do you run a southern blot?
extract DNA, do a restriction digest and treat to make single stranded - transfer to a membrane and hyb a radioactive probe to it
what is the lower detection limit of DNA by southern
0.5 pg
what are drawbacks to southerns?
takes a long time, radioactivity, only one probe at a time
how do you FISH?
arrest cells in metaphase, hybridize with fluorescently labeled conjugates, take pics
drawback to FISH?
hyb artifacts
how does Crispr-Cas knockout genes?
you cleave at a specific, targeted location, which is then repaired by NHEJ which 2/3 of time sticks in an indel, messing up the reading frame and knocking out the gene
what type of macromolecule does a northern blot assay for?
RNA
what is a gibson assembly?
quick way to stitch together multiple pieces of DNA - requires linearization of vector, can be annoying for big or repetitive inserts
why would you use NET-seq?
to detect nascent, actively transcribing RNAs
why would you use RACE-seq?
to sequence 3’ and 5’ UTRs of cDNA ends through anchored PCR
what is polysome analysis?
you separate mRNAs into actively translating and non-translating fractions depending on polysome association
what can you use ribo-seq to look at?
determine translation initiation/termination and alternative ORF sites, or sites of ribosome pausing/slippage
