Choose Your Vector
Insert Size: Small
Uptake: Transformation
General Usage: Genomic library, subcloning, shuttle plasmid for expression in eukaryotes
Plasmid
Cloning
1. Goal/Purpose: gene of interest
2. Vector Type: selection method for obtaining cells
3. Insert DNA: isolated fragment/genomic DNA or cDNA
4. restriction enzyme or if library blot/probe for screening method
Genomic Libraries
A collection of clones that contains all the DNA sequences of an organism's genome (contains coding and noncoding segments)
Insert Size: Medium
Uptake: transduction (infection)
General uses: subcloning, random mutagenesis
Phage
Hybridization
1. colonies on the plate, each colonies had a recombinant plasmid with a different insert
2. colony blot: lay filter on surface , some cells form each colony will stick
3. Probe: radioactivity labeled DNA probe
4. imprint: DNA fixed on surface
5. autoradiography
Complementary DNA (cDNA) Libraries
collection of cloned cDNA sequences (represents only the genes being expressed in cells at the time the library was made)
Insert Size: Large
Uptake: Transduction (infection)
General uses: Large-scale sequencing/mapping, chromosome walking
Cosmid
Functional Complementation
1. plasmid library
2. transform yeast selecting for plasmid marker
3. Yeast transformation
4. Select or screen for gene of interest
Repair by NHEJ
sticks ends together, prone to making indels (nonhomologous joining)
Insert Size: Huge
Uptake: Transformation
General Uses: Whole genome sequencing, low copy number
Bacterial artificial chromosomes (BAC)
Conditional Knockouts
1. Insert sequences called LoxP sites into the targeting vector on either side of the gene of interest
2. Crossed the other mice with a germline containing the Cre gene, which encodes Cre recombinase, recombines them and deletes the gene between the LoxP sequence
Repair by HDR
uses DNA you put in cell to replace damaged portion (homologous direct repair)
Insert Size: Gigantic
Uptake Transformation
General Uses: Genome Sequencing, have telomeres at each end, ORI, and centromere
Yeast Artificial Chromosome (YAC)
Knockouts
1. DNA sequence for gene of interest must be known
2. Target vector is constructed to create a segment of DNA that can be introduced to the cells (reporter genes produce KO or transgenic organisms that are easy to detect)
3. Undergoes homologous recombination with the gene of interest (the target gene) (recombination disrupts or replaces the gene of interest, therefore rendering it nonfunctional)
PAM sequences
help bacteria distinguish bacterial DNA from foreign DNA and are not part of the CRISPR locus (protospacer adjacent motif)