Lab 6

The number of colonies on the media without antibiotics tells us how much…

bacteria are present in the saline wash

How do you find the proportion of the population that are resistant to that antibiotic?

number of colonies on the media with antibiotics/ number of colonies on the plates without antibiotics

If the proportion of the population that are resistant to that antibiotic is less than 100, what does that mean?

Correct

If the proportion of the population that are resistant to that antibiotic is more than 100 what does that mean?

due to pipetting mistakes

Ampicillin

beta-lactam that targets the cell wall

Sulfonamides

a class drugs that are bacteriostatic, that is they slow bacterial growth by targeting metabolism, but don’t kill the cells

Streptomycin and Tetracycline are…

2 different classes of drugs that both target protein synthesis by binding to the ribosome

How do you test for resistance with multiple antibiotics?

• Amp + Sul

• Amp + Strep or Amp + Tet

Where do you put your plates after phenotyping?

Into the incubator 37C

In DNA purification, what does the centrifuge do?

Remove the cells from the media

In DNA purification, what does a spectrophotometry do?

Measure the amount and purity of DNA that we produced

Creation of lysate

• Mechanical disruption is used to physically break apart the bacterial cells

• Sterilized silica beads are larger than bacterial cells and smash them to bits when the tubes are shaken in the vortexer

Creation of lysate - FINAL PRODUCT

Soup of bacterial bits, which includes DNA and all the contents of the cell

Clearing of lysate

DNA is in the solution with other cell bits

How is protein precipitated in the clearing of lysate stage?

Ammonium sulfate or trichloroacetic acid

In the clearing of lysate stage, what does the buffer do?

• ‘Proprietary buffer’ (IPS)

• Its and larger protein aggregates can be separated from the DNA solution by passing it through a paper filter

• Contains high concentrations of EDTA, which chelates cations from enzymes and helps to prevent the degradation of DNA by a class of enzymes called DNAase, which will break down DNA if left uninhibited

What does binding to the matrix allow us to do?

Clean the DNA by passing wash solutions over the beads

Binding to the matrix uses a proprietary buffer…

• SB

• Phosphate backbone of DNA is pos and silica beads are neg → buffer contains high levels of chaotropic salts (extra abundance of pos charge enhances the binding of DNA to silica, when solution passes, DNA binds to the silica beads and is immobilized)

Washing

• DNA washed with buffers that have alc

• DNA isn’t soluble in high percentage alc solutions and thus stays put on the silica

• Helps to remove any protein and cell bit contaminants that might have been trapped in the column

Elution

• GetDNA off the column

• DNA is readily freed off the column silica by water or neutral pH buffers such as TE, which also contains EDTA that will help to guard our precious samples from degradative enzymes

If Solution SL has precipitated, heat at…

55C for 5-10 mins

Perform all centrifugation at ___ temp

room (15-25C)

Secure PowerBead Tubes horizontally using the Vortex Adapter with rubber bands. Vortex at maximum speed for 10 mins, This step creates the…

Combined chemical/mechanical lysis conditions required to release desired nucleic acids from microbial cells

Solution IRS

• Contains a reagent to precipitate non-DNA organic and inorganic material, including cell debris and proteins

• Important to remove contaminating organic and inorganic