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
what is CLIP?
cross linking immunoprecipitation, used to map protein binding sites to RNA through either UV crosslinking, an antibody conjugated to biotin or HRP, magnetic beads, etc
how can you tell how much protein you have in your sample?
colorimetric methods, bradford/coomassie, or UV absorbance spec or western blots if you have an antibody
how do you overcome transient interactions in protein co-immunoprecipitation?
one way is crosslinking, another is proximity dependent labeling (Bio-ID). You modify protein of interest so it contains a biotin ligase - this ligase sticks proteins onto any other proteins within its reach
T/F. Flow cytometry can provide you information about protein localization
False, immunofluorescence and live cell imaging will do better.
