week 3 Flashcards
control gRNA vs smo gRNA
guides cas9 to no genes. so none of them will be cut/have indels
guides cas9 to smo gene, giving it indels
T7 endonuclease I (T7EI) nuclease assay
detects mismatches in DNA that results from denaturing and reannealing PCR products (amplified smo gene). when reannealed, this could result in wild type or indels
heteroduplex
DNA (formed after T7EI) assay that is in between wildtype and crispr-mutated DNA
T7 endonuclease I
recognizes and cleaves nonperfectly matched DNA
What are the three types of dna pcr is amplifying?
wild type gene, gene with insertions, and gene with deletions
hairpin loop
loop formed between DNA that is amplified during PCR (wildtype (normal # of bps), insertion (extra bps), and deletion (fewer bps)). these form when, after denaturation, strand anneals with a strand of a diff “type” (ex: WT anneals with insertion strand). insertion has extra bps so they won’t perfectly complement WT and will instead form the loop
T or F: if only a single base in WT, insertion, or deletion DNA strand changes, the strands can reanneal with each other without any hairpin loops forming
true. thus, we don’t notice single base changes and the actual number of base changes could be higher than we think
tide sequencing primer: what does it do?
it sequences the bases in the template strand until it reaches smo #2 guide site (one of the cut sites). once it reaches here, it tells us the percent of indels/nonindels in this area, and thus tells us how efficient the smo guide was in comparison to the T7 assay
t7 assay on agarose gel tells us…
agarose gel contains bands. some bands have the full length PCR product, some only have partial product (because the DNA had a hairpin loop that was cut by T7). The fluorescence of partial bands/full length DNA band tells us the number of indels (more indels=more partial bands=more fluorescence of those)
SYBR safe stain
used for DNA fluorescence
positive electrode is at top/bottom of well. negative electrode is at top/bottom of gel.
bottom; top
true or false: running buffer contains bromophenol blue and 10% glycerol.
false. this makes up loading buffer, which we use so DNA sinks in well (since glycerol is heavier than water, which is what running buffer is partly made of)
running buffer: what it does and what it’s made of
sets up pos and neg side of gel so current can run through it. made of Tris, Acetic Acid, and EDTA (TAE)
… underestimates the amount of edits caused by cas9, so we will use … to confirm
T7 nuclease; base sequencing
chaotropic salt
used for dna purification: allows DNA to bind to silica. the proteins/primers on dna are washed off so we’re just left with dna
pure DNA has optical density of
260:280 or 1.8
DNA has max. absorbance at
260 nm
