controls for transformation

purpose of selective markers

  1. eliminate cells without plasmid

  2. identify cells without recombinant DNA

  • antibiotic resistance gene: cells that did not take up the plasmid are not resistant to ampicillin and do not form colonies on media containing this antibiotic

  • lacZ gene: cells that took up plasmids without DNA fragments have functional lacZ genes, hence are able to metabolize X-gal, and turn blue on media that contain X-gal

controls are experiments run alongside the test sample to ensure that

  • all test subjects/components/reagents/devices used in the experiment are functional

  • serve as a benchmark so we know how different the test sample is from the benchmark

Positive control: test conducted using a sample that is known to show positive result

Negative control: test conducted using a sample that is known to show a negative result

Purpose of transformation - insert recombinant plasmid into host cell

steps for transformation: heat shock at 42ºC for 1 min

what needs to be in the agar to select for transformed cells: antibiotics, X-gal

what to expect after transformation

Blue colonies - bacteria with plasmid

White colonies - bacteria with recombinant plasmid

  • if spread plate contains only has white colonies: ampicillin not functional, bacteria culture contaminated, X-gal not working

Need to count the ratio of blue to white colonies

Negative control for transformation

  • competent cells + buffer, spread on agar with antibiotic + X-gal

  • expect no colonies

  • checks if antibiotic in the agar is functional

  • checks if any contamination occurs

  • mistakes result in colonies

  • purpose: to show that cells are competent and can take up the intact plasmids

No colonies formed (possibilities)

  • LB plate not prepared properly or not suitable for bacterial growth - incorrect pH/nutrient/antibiotic

  • the cells were not competent to take in plasmid

  • all competent cells died even before experiment

  • transformation steps were not correctly done

Positive control for transformation

  • competent cells + undigested plasmid, spread on agar with antibiotic + X-gal

  • expect only blue colonies

  • checks if transformation steps are successful

  • checks if the cells are competent

  • checks if competent cells are healthy/dead

  • checks if the medium/agar is well prepared (suitable pH, nutrient content, X-gal, etc)

  • any mistake results in plate full of white colonies / no colony

  • purpose: to show that non-transformed cells are susceptible to the antibiotic

    • bcs in the neg control they would die to the antibiotic to this is to check if they are actly dying because of the antibiotic - check if they are actly working or jsut bcs there are no cells

how to ans these qns: biological molecule (what it has) - biochemical action (what will happen) - observations (what we see)

example:

negative control

  • antibiotic will kill cells, plasmids that contain antibiotic resistant gene would not be killed hence those cells with the recombinant plasmid can express enzymes to break down the antibiotic and survive in the presence of the antibiotic and colonies are formed