Molecular Techniques I: DNA Flashcards
Types of dna techniques
“to clone Paul C.Rxn from down South, go to the Library and return with the Sequence”
Cloning
PCR
Southern Blot
DNA library
Sequencing
Requirements for cloning
Plasmid
Eukaryotic DNA
Restriction enzymes (endonuclease)
Ligase
Cloning procedure
Plasmid requirements
Origin of replication
Selectable marker
Restriction site
Gimme: what do you use for foreign dna that’s more than 20kb?
Bacterial Artificial Chromosome
Yeast Artificial Chromosome
Clustered restriction sites are called
Multi-cloning regions
How do restriction enzymes work?
Cut at 4-6bp sequences (aka restriction sites) that are palindromic
Possible ligation products after cloning
Transformation requirements
Competent cells (electrocompetent or heat-shock based)
Electroporation/Heat shock
Antibiotic selection
Colony screening
Transformation procedure
Colony screening types
Blue white screening
Hybridization
(Colony PCR w/ flanking primers and gel)
Blue white screening procedure
Hybridization procedure for colony screening
How do you make a DNA/RNA probe?
Find 6-7 aas in a row in a region with the least degeneracy
Total probe size = 18-21 nucleotides long
Radiolabel 5’ end
Polymerase chain reaction procedure
Calculation of copy number after PCR
2^n where n = # ds strands generated/cycle number +1
Describe how you would use PCR to identify mutations in known genes using the CFTR gene as an example
(check sheet for answer)
Need to design primers that flank the normal region of the gene and the deleted/mutated region of the gene, amplify DNA then run a gel
If HM normal, gel will show ONE large band
if HT, gel will show 2 bands (one large, one small)
if HM mutant, gel will show ONE small band (mutated region)
Southern Blotting procedure
SNP-based Restriction fragment length polymorphism analysis
SNP abolishes restriction enzyme site
STR based RLFP analysis concept
DNA library types
cDNA library construction process
Genomic and cdna
Sanger sequencing (old school) concept and procedure
Based on chain termination (via ddNTPS) and 4 separate reaction mixtures
Sanger sequencing (new school) procedure
(still chain termination) One reaction and capillary gel. Signal read by laser since ddNTPs have flourophore
Next Generation sequencing procedure
Why is it called “massively parallel?”
Ion torrent Next Gen Platform
Reverse termination/Bridge PCR (Illumina) NExt Gen
Bridge PCR for Illumina
