Molecular bio Wk 5

What are the two transformation methods?

-chemical transformation -electrical transformation

What is chemical transformation?

-divalent cations cause the cell membrane to become more permeable -positive charge of divalent cations attracts negative charges from DNA and lipids in cell membrane -cells are treated with CaCl2 prior to exposure to DNA

What is electrical tranformation?

-aka electroporation -e.coli cells are made competent for electroporation: cells grown washed in low ionic strength solution, resuspended in low ionic strength solution containing glycerol -electrocompetent cells can be stored at -80C for later use or used directly for electroporation to introduce DNA

What does Electroporation involve?

-mixing electrocompetent cells with ligation mix or plasmid DNA -mixture added to an electroporation cuvette -high voltage electrical pulse passes through cells cause: disruption to cell membrane, temporary pores in cell membrane, uptake of DNA -recovery and plating similar to chemical transformation

What are different selection methods to detect recombinant DNA?

-antibiotic resistance markers -dual antibiotic resistance

What are different screening methods to detect recombinant DNA?

-blue/white screening -colony PCR -restriction digest

What is antibiotic resistance markers?

plasmid contains a gene for antibiotic resistance. only bacteria with the plasmid survive on antibiotic plates

What is Dual Antibiotic Resistance?

inserting DNA into one resistance gene disrupts its function. colonies that are resistant to amplicon but not tetracycline have the insert

What is blue/white screening (LacZ)?

-uses the lacZ gene that produces b-galactosidase -plasmids with no insert = functional LacZ =blue colonies (on X-gal) -plasmids with insert = disrupted LacZ = white colonies (insert = recombinant)

What is colony PCR?

rapidly checks for presence and size of insert by amplifying DNA directly from colonies

What is restriction Digest?

isolate plasmid DNA and cut with enzymes to verify insert size and presence via gel electrophoresis

What are the steps to plasmid DNA isolation?

1. resuspension

2. lysis

3. neutralisation

4. centrifugation

5. purification

What is resuspension?

bacteria are pelleted and resuspended in buffer

What is Lysis?

alkaline solution breaks open cells and denatures DNA

What is Neutralisation?

acidic buffer causes chromosomal DNA and proteins to precipitate

What is centrifugation?

plasmid DNA stays in solution; debris is pelleted

What is purification?

DNA is cleaned up using silica columns or alcohol precipitation

What are different strategies used for cloning DNA molecules?

-using restriction enzymes -PCR-based cloning -TA cloning

How to use restriction enzymes

-DNA is cut with restriction enzymes and ligated into similarly cut plasmids -directional cloning uses two different enzymes to control insert orientation

How to use PCR-based cloning?

-primers can be designed to add restriction sites to ends of PCR products -useful when the insert DNA doesn't naturally contain the needed restriction sites

How to use TA cloning?

PCR products with A-overhangs can be cloned into T-overhang vectors (no need for restriction digestion

What are the different methods for delivering recombinant DNA into Eukaryotic cells?

-physical methods -chemical methods -viral vectors

What are physical methods for delivering recombinant DNA into Eukaryotic cells?

-electroporation -microinjection -gene gun (biolistics)

What is Electroporation (physical methods)?

electric shock creates pores in the membrane

What is Microinjection (physical methods)?

direct injection into nucleus or cytoplasm using fine needles

What is gene gun - biolistics (physical methods)

DNA-coated gold or tungsten particles shot into cells

What are chemical methods for delivering recombinant DNA into Eukaryotic cells?

-calcium phosphate transfection -lipofection

What is Calcium Phosphate Transfection?

forms DNA precipitates that are taken up by cells

What is Lipofection?

DNA mixed with liposomes fuses with cell membranes

What are the viral vectors?

use modified viruses to deliver genes efficiently into dividing or non-dividing eukaryotic cells