LECTURE 02 - Syllabus
LECTURE 02 - Spectrometer
● A = ecl
○ Absorbance
○ Extinction coefficient
○ Length of light
● Graphing - absorbance vs concentration
○ Slope = extinction coefficient
● Pipettes
○ Blow out/serological - two circles at top, goes to 9mL
○ To deliver/Mohr - no ring at top, goes to 10 mL
○ Volumetric/transfer pipette - exact amount
● DNA - OD260
● Protein - OD280
● Growth curve - lag phase, log phase, stationary phase, death phase
○ High turbidity = high absorbance = low transmittance = many cells = either dead or alive
LECTURE 03 - cell growth
● Bacteriostatic - does not kill organisms, inhibits reproduction
● Bactericidal - kills organisms by inhibiting cell wall synthesis
● Colony counting
○ Less than 20 = TFTC
○ More than 300 = TMTC
LECTURE 04 - molecular cloning
● 3 distinct properties of a vector
○ Origin of replication - able to replicate independently of host
○ Restriction enzyme site - allow target foreign DNA to enter
○ Selectable marker gene - select for cells that can take up vector
● Competent - cell capable of taking up vector
● Transformant - host that has taken up vector
● Recombinant vector - foreign DNA inserted
LECTURE 05 - DNA sequences
● 5’ overhang has a short 5’ end
● 3’ overhang has a short 3’ end
● Blunt ends has equal length cut fragments
LECTURE 06 - Restriction Maps
● Anchor sites - places the enzyme cuts the DNA into fragments, sites
○ Second anchor site - used for another enzyme or to find double digests
● DNA fragments
○ Large - nicked
○ Medium - linear
○ Smallest - supercoiled
● PROCEDURE - RESTRICTION DIGEST
○ Each digest contains
■ Plasmid (recipient DNA holder)
■ Restriction buffer (sink in gel and colour)
■ Sterile water (volume)
■ Restriction enzymes
● EcoRI
● HindIII
● KpnI
● BamHI
○ Tap to mix / briefly spin
○ Incubate in 37C-45C for 35min-45min
○ Already have Anza Red Dye, add bromophenol blue dye if necessary
■ Loading dye and loading buffer will cause restriction enzymes to cut incorrectly
○ Leave for next day
LECTURE 02 - Spectrometer
● A = ecl
○ Absorbance
○ Extinction coefficient
○ Length of light
● Graphing - absorbance vs concentration
○ Slope = extinction coefficient
● Pipettes
○ Blow out/serological - two circles at top, goes to 9mL
○ To deliver/Mohr - no ring at top, goes to 10 mL
○ Volumetric/transfer pipette - exact amount
● DNA - OD260
● Protein - OD280
● Growth curve - lag phase, log phase, stationary phase, death phase
○ High turbidity = high absorbance = low transmittance = many cells = either dead or alive
LECTURE 03 - cell growth
● Bacteriostatic - does not kill organisms, inhibits reproduction
● Bactericidal - kills organisms by inhibiting cell wall synthesis
● Colony counting
○ Less than 20 = TFTC
○ More than 300 = TMTC
LECTURE 04 - molecular cloning
● 3 distinct properties of a vector
○ Origin of replication - able to replicate independently of host
○ Restriction enzyme site - allow target foreign DNA to enter
○ Selectable marker gene - select for cells that can take up vector
● Competent - cell capable of taking up vector
● Transformant - host that has taken up vector
● Recombinant vector - foreign DNA inserted
LECTURE 05 - DNA sequences
● 5’ overhang has a short 5’ end
● 3’ overhang has a short 3’ end
● Blunt ends has equal length cut fragments
LECTURE 06 - Restriction Maps
● Anchor sites - places the enzyme cuts the DNA into fragments, sites
○ Second anchor site - used for another enzyme or to find double digests
● DNA fragments
○ Large - nicked
○ Medium - linear
○ Smallest - supercoiled
● PROCEDURE - RESTRICTION DIGEST
○ Each digest contains
■ Plasmid (recipient DNA holder)
■ Restriction buffer (sink in gel and colour)
■ Sterile water (volume)
■ Restriction enzymes
● EcoRI
● HindIII
● KpnI
● BamHI
○ Tap to mix / briefly spin
○ Incubate in 37C-45C for 35min-45min
○ Already have Anza Red Dye, add bromophenol blue dye if necessary
■ Loading dye and loading buffer will cause restriction enzymes to cut incorrectly
○ Leave for next day
