Lab7+8 Flashcards
How do you pick the right promoter
RNA poly II for cas 9
RNA poly III for driving gRNA
gRNA should have G at 5’ end for U6 OR A at 5’ for U3
What should U3 promoters have
TATA box
promoter specific to monocot
upsteam sequence element
What can improve efficacy of gene editing
plant species specific promoters
How do you identify and annotate plant U3 promoter
BLAST to find small nuclear RNA U3 gene
retreive 1000bp upstream
idenify and annotate them
validate efffiency
What is guide DNA
- specific 17-24 nuc sequences used for cas nucelase
What is PAM
protospacer adjacent motif, short sequences near gRNA for cas nucelase to cut
What are the requirment for a good guide
20 bp
GC 40-60
in coding sequence
zero mismatch tolerance
high activty and specific score
targets 1st exon and all homelogs
How do you decide amongst gRNAs
- mismatchs hould follow PAM, at least two should be within PAM proximal regional
- the global similarity to target should be small
- the mismatches should be consequtive or less than 4 bp apart
What are the pros os Bsai
- has a defnite recongition site and you can design own overhangs
- Recognition site is external to cleavage site as a result there are no BsaI sites in the finished product
What are methods to screen transgenics
sanger sequenction
restriction digestion
deep sequencing
T7E1 Essay
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FOR RNA Extraction what do you need?
BME
Buffer RLT
Buffer RLC
QIA Shredder
Ethanol
RNeasy Mini Spin Column
For RNA Extraction, what do these do?
Beta mercaptoethanol: reducing agent to denature RNases, removes disuflide bonds
Buffer RLT + RLC: guanidine thiocyanate and guanidine hydrochloride
RLT: higher cell disruption + denatures
RLC: milky endosperm , removes insoluable material
QIA shredderL homogenisation, reduce viscorty
Ethanol: promotes selective binding of RNA to RNeasy membrane
RNeasy Spin column: Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNAse-free water
