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How do you get DNA out of the cell?
- Mechanically- Blender
- Chemically- enzymes and detergents
What are enzymes that glue fragments of DNAcalled?
What do blunt ends require to keep them stably glued?
T4 DNA ligase (viral)
How do we separate DNA fragments?
Restriction Buffer provides optimal conditions with NaCl, Tris-HCl and Mg2+. What do each of these do?
- NaCl- correct ionic strength
- Tris-HCl- proper pH
- Mg2+ - enzyme co-factor
What is a restriction map?
A diagram that shows the location of RE cut site on pieces of DNA sequence
What is a vector?
It is a DNA vehicle to deliver DNA into a host cell
What are the requirements to work with plasmids?
- Detect presence of the vector
- Direct selection method for cells
- Insert genes into vector
- Detect the presence of an inserted gene
What is transformation?
Putting the cloned genes into bacteria
What is the charge of nucleic acids?
What is southern blotting used for?
Locate a particular gene
What is PCR used for?
- Clinical diagnosis
- Genetic analysis
- Genetic engineering
- Forensic analysis
What do we need for PCR?
- Primer (forward and reverse)
- PCR machine
What are the three steps of PCR?
What do we use degenerate primers to find?
DNA sequence of an organism
What are the 2 major steps required for purifying DNA?
- Centrifugation- separation by density
- Chemical Extraction
What are the two types of restriction enzymes?
- Type I- cuts DNA faraway
- Type II- middle of recognition site (blunt and sticky end)