week 4 Flashcards
what is imageJ used for
calculating % of indels caused by t7 endonuclease by circling fragmented bands, measuring their fluorescence, and using this value to calculate % of indels
DNA sequencing
determines the order of the nucleotides that make up the DNA molecule of interest
specific type of dna sequencing and what it’s used for
dideoxy (sanger) method (aka chain termination method): used to sequence small genes like those used in plasmids/PCR
dNTP vs ddNTP
Deoxy-ribo-Nuceloside-TriPhosphate: nucleotide with nitrogenous base, 3 phosphate groups, and ribose with OH group at 3’ end.
Di-Deoxy-ribo-Nuceloside-TriPhosphate: nucleotide with nitrogenous base, 3 phosphate groups, and ribose with no OH group at 3’ end.
how does the chain termination method work?
get rid of the OH group at the 3’ end of the nucleotide so the next nucleotide can’t be added. so the chain of DNA is stopped
TIDE primer adds to the …’ end of DNA during chain termination
3
for chain termination reaction, we add lots of … and a few …
dNTPs (regular nucleotides that will add to primer)
ddNTPs (chain terminating nucleotides that are fluorescently marked and will add to primer)
true or false: TIDE primer has to bind to T nucleotide in the guide sites (DNA sites that guide RNA will bring cas to)
true. if there is an indel at this site, TIDE primer might not bind to T (bc it’s not there). so the fluorescence readings will be scattered
why are we not using a Smo protein for western blot that is endogenous/already present in drosophila?
smo protein is large/degrades easily
true or false: we’re tagging smo gene with HA-tag
true. we’re inserting smo into plasmid where it will get a HA-tag
ACT5.1 promoter is constitutive/inducible
MT promoter is constitutive/inducible
constitutive- always going
induced with copper
What are we putting in plasmid, and what promoter will be used for each?
Control (nothing)- actin/ACT5.1
Hh- ACT5.1
Smo with HA tag- ACT5.1
Drice with V5 tag- MT
Dcp1 with V5 tag- MT
Dark with V5 tag- MT
HA tag: how many AA, where it’s found, which terminal its on
V5 tag: how many AA, where it’s found, which terminal its on
9 amino acids, found in flu virus’ hemagglutinin protien, on c terminal of smo
14 amino acids, found in simian virus’ 5 RNA polymerase protein, on n terminal of caspase
what are we using plasmids to insert?
Hh, smo-HA or caspase-V5, and
what is TRN reagent coating?
RNA (controlguide and smoguide) and DNA (smo-ha plasmid dna and Hh plasmid dna)… and maybe caspase idk
