Lecture 15 - Cloning and gene expression Flashcards
Define cloning and examples of what it’s used for
- isolating genes from genome by amplifying/cloning DNA
- to characterise and study genes = mapping, sequencing, manipulation
What are the 2 general processes of cloning depending on known info?
- No info (phenotype only) = isolate, fragment, clone into vectors, library, screen
- Info (sequence similarity) = amplify, clone into vector
Compare and contrast random vs specific cloning
- Random = unknown, use RE fragments, don’t need known, need multiple REs in library + screening
- Specific = known, use PCR fragments, easier and less work, need known sequence
Briefly describe the 6 steps in random cloning
- o High quality high weight sample
- Fragment donor into vector with same RE
- Ligate with T4 DNA ligase
- Transfer to E coli host and amplify
- Screen for recombo plasmid
- Purify and analyse
Explain chemical transformation
- Small DNA fragments
- log phase E coli in CaCl2 so cations interact with -ve E coli membrane (lipid A) = destabilise to take up
- 10^6 to 10^8 colonies
Explain electroporation transformation
- Big DNA fragments
- log phase E coli in water + electric current to force uptake
- 10^8 to 10^10 colonies
What are plasmid cloning vectors and what do they need to have?
- Small plasmids
- Selectable marker = resistance
- Multiple cloning site (MCS) = multiple RE sites
- oriV + high copy number of plasmids = better amplification
What is agarose and what is the buffer used?
- polymer of repeating di-galactan units
- pH8 buffer of tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE)
What are the 2 fluorescent DNA binding agents? How does DNA sink to the bottom of the loading wells?
- ethidium bromide or SYBR green
- loading buffer + sucrose to sink
Explain the role of T4 DNA ligase - origin, function, requires
- from T4 phage
- phosphodiester bond between vector and donor DNA
- ATP, Mg2+, optimal pH, 16C incubation
Explain the role of alkaline phosphatase - origin, function
- from bacteria, shrimp, calf
- stop self-ligation of sticky ends by removing 5’ phosphate so only vector 5’ phosphate is used
- removed before ligation
Explain how DNA ends are made/are compatible for DNA ligation
- Compatible DNA ends = blunt or sticky but blunt better bc no bpairing
- T4 DNA ligase exonuclease activity like Klenow fragment to fill in/remove overhangs
When is blue-white screening used and what series/plasmid is used?
- When no phenotype
- pUC series = pBR322 replaces TetR with lacZ of beta-galactosidase
Briefly explain the 4 steps to blue white screening
- Digest foreign and pUC18 with Sal1
- Ligate
- Transform into E coli
- Plate with ampicillin, IPTG and Xgal
What are the 3 possible results of blue white screening?
- Blue colonies = self-ligation (no recombo)
- White colonies = recombo (interrupted lacZ/X-gal)
- No colonies = didn’t take up recombo plasmid with resistance
Where is lacZ originally from and how does it work in blue white screening?
- E coli lac operon = P, LacZYA
- lacZ = beta galactosidase cleaves lactose to galactose + glucose (repressed)
- synthetic lacZ = X-gal cleaved = blue precipitate
- lacZ omega on host + lacZ alpha on vector coexpressed and joined = lacZ
- Coexpression controlled by synthetic isopropyl-beta-thiogalactoside (IPTG) (normally LacI)
- recombo gene interrupts lacZ/X-gal
