Bacterial and yeast techniques Flashcards
What is bacterial transformation?
- forcing competent E. coli to take plasmid into cell
What are competent bacterial cells?
- chemically treated w/ Rb/Ca/Mn chloride
- facilitates attachment of DNA to cell membrane
What is the purpose of heat shocking cells in bacterial transformation?
- opens membrane pores to allow plasmid to enter cell
Why do cell need time to recover after heat shock in bacterial transformation?
- allow exp of antibiotic resistance genes successfully transformed w/ plasmids
How is selection performed after bacterial transformation?
- transformation mixture plated on selective agar approp to plasmid inserted
- eg. Ampicillin plates select for cells that have integrated plasmid containing AmpR gene
- transformed cells = survive
- non-transformed cells = die
How is transformation efficiency calc?
- CFU / ug pUC19 plasmid
- higher then efficiency, the higher the quality of competent cells originally
What factors can effect transformation efficiency?
- temp
- DNA sample
- heat shock (temp and time)
- media used for recovery step
- plasticware used
- recovery time
- user handling/error
What is auxotrophy?
- the inability of an organism to synthesise a particular organic compound required for growth
What are the general steps of yeast transformation?
- prepare competent cells
- incubate competent cells with DNA
- heat shock
- selection using appropriate agar plates
How should cultures be grown for metabolic processes like transformation?
- cell cultures should be harvested when in the mid-log phase of growth (S.cerevisiae OD600 0.4-0.9)
- these cells are rapidly dividing and metabolically active
- harvesting in other growth stages significantly affects experimental outcome
What is the purpose of PEG 4000 in yeast transformations?
- thought to help bring DNA close to cell membrane
- stabilises cell membrane
- acts as molecular crowder, effectively increasing the DNA conc around cells
What is the role of salmon sperm DNA in yeast transformations?
- acts as carrier molecule, to aid uptake of plasmid DNA
- target for nucleases, reduces chance of plasmid degradation
What are some common mistakes in yeast transformations?
- incorrect/old PEG solutions
- ssDNA not prepared correctly, must be boiled and quickly chilled to maintain its single-stranded DNA
- incorrect selection plates
How does T4 DNA ligase buffer work?
- contains ATP, essential cofactor for the reaction
- very heat labile (defrost buffer on ice)
- vortex buffer well as ATP can precipitate out of solution
Components of YPAD broth/agar and purpose?
- yeast extract = nutrient source
- peptone = peptic (enzymatic) digestion of protein and nitrogen/amino acid source
- adenine = req for ade2-1 yeast, colonies turn red if absent
- dextrose = energy source
