CRISPR CAS SYSTEM
What is crispr cas 9 system made up of ? What is it used for?
Guide RNA and Cas 9 Protein
Genetic modifications of organisms (GMOs)
what does PCR stand for?
Polymerase chain reaction
Forward Genetics
observing a phenotype to find the genotype causing it
What is the purpose of electrophoresis and why is it useful to us ?
Purpose: technique used to separate DNA fragments by size
useful to verify that the PCR worked and that there was product to be sequenced.
- having product refers to having a working concentration.
Where did Crispr cas 9 originate from? Why?
Bacteria developed it as a form of an immune system
what is PCR used for?
used to amplify DNA sequences to a working concentration
reverse genetics
observing the genotype to see the phenotype
useful to see the purpose of a gene
ex: get a gene, alter it, and see the effect it has on phenotype
Principle that allows electrophoresis to work?
DNA has a negative charge that flows in one direction (positive direction towards cathode of electrode)
The guide RNA (CRISPR)finds a target sequence and hybridizes with its opposing DNA sequence to create an R loop. The R loop stabilizes. This is a signal for Cas 9 to start cutting and make a double stranded break.
why is PCR useful to us? What is working concentration?
PCR can help us see a small gene or DNA sequence as a working concentration by amplifying it.
Working concentration is a high concentration of something that is useful to do other experiments .
What are the different ranges of micropipettes?
P2 - 20
P20-200
P100-1000
T/F- Size of DNA fragments does not matter because charge to mass ratio will always be the same
true! this is the reason why you get bands on the gel regardless of size
What happens after the double stranded break done by cas 9 ?
DNA repair through two pathways:
1. Homology Dependent Repair
2. Nonhomologous Ending Joining
Provide temperatures for each
1. denature - Hydrogen bonds are broken in double stranded duplex; separate 2 DNA strands
temperature : 95ºC (high temp to denature)
2. Annealing - primer anneals to target sequence
temperature: 55ºC (solution has to cool down so primer can bind
3. Elongation (extension) - extending primer into a new strand of DNA
temperature: 72ºC (TAQ polymerase is most catalytically efficient)
if you need to pipette a 200 microliter solution what micropipette do you use? Why?
P20-200 should be used because there is a smaller margin of error compared to a micropipette that has a larger range (ex: P100-1000).
T/F- Bigger fragments travel slower because of the sieve of the agragose
true! smaller fragments travel easier than bigger ones
What is nonhomologous ending joining ? What are indels?
DNA repair pathway that is error prone and can create indels.
Indels - frameshift mutations (insertions or deletions of nucleotides)
Priniciple that makes 1 strand of DNA turn into the working concentration that is needed?
Exponential
How do you read gel ?
*know how to read a nucleotide sequence to get a gel
* know how to read a gel to get a nucleotide sequence
*Always include RUN and READ
*ALWAYS include 5' to 3'
how is DNA visualized? What components of it allow it to visualize DNA?
1. CyberSAFE
2. ait is a DNA intercalator which means it loves the hydrophobic regions (bases) of DNA. It has a high affinity to DNA
2b. it has fluorescence properties. When you blast it with UV light or Blue light, it absorbs high energy light and emits it as lower energy wavelengths