Flashcards in Principles Of Cloning Deck (20):
Methods of transfer of genetic material
Why do we clone?
- reproduction if specific DNA fragments in large amounts
- stable propagation of DNA sequences
- allows DNA to be studied, manipulated and expressed
Applications of cloning
- construction of genetic libraries
- production of scarce proteins
- mass production of vaccines
- gene transfer
What is the restriction modification system.
Host DNA is methylated to protect it from cleavage by REs
What factors are Re groups based on?
- enzyme cofactor requirements
- nature if target sequence
- position of DNA cleavage sites relative to target sequence
How does DNA ligase work
Forms phosphodiester binds between 5' p and 3' OH
What is cDNA used for?
- expression studies
- gene structure and function studies
4 strategies for cloning PCR products
- blunt end PCR cloning
- restriction site cloning
- T/A cloning
What must cloning vectors contain
- origin if replication
- selectable marker
- multiple cloning site
What are expression vectors
Allow a cloned segment if DNA to be translated into a protein inside a cell
What must expression vectors contain?
- in vivo promoter
- antibiotic selection
- sequencing primers
What is a linker.
Small dsDNA that contains a new RE site
What is directional cloning?
Cut both vector and insert with 2 different REs
Low background of non-recombinants
What does AP do?
Removes 5' phosphatase from vectors to prevent self-ligation
NB in blunt-end cloning
How to calculate molar ratios
(Size of insert in kb/size of vector in kb) x ng of vector = ng of insert needed for a 1:1 ratio
The ability of a cell to take up extracellular naked DNA from its environment
How to increase competency
High ca2+ conc makes small holes in the cell membrane
Number of colonies per ml/DNA conc transformed
Steps to isolate plasmid DNA
- re suspend in solution 1 (tris-HCl buffer, EDTA, Glucose)
- add solution 2 (SDS, Naoh)
- add solution 3 (potassium acetate, acetic acid)
- precipitate plasmid DNA with isopropanol
- restriction mapping