Team:Waterloo/Experiments

Escherichia coli
Restriction Enzyme Digest Inoculating liquid cultures Inoculating liquid cultures
Saccharomyces cerevisae
Fluorescence
Plasmid Miniprep
  1. Restriction enzyme digest is a process in which restriction endonuclease identify a restriction site within the DNA sequence and cleave them, resulting in overhang or blunt ends. Type II restriction enzymes, such as EcoRI and PstI, allows you to cut at specific palindromic sites on multiple target DNA and attach them to one another by ligation of the complementary ends.
  2. Calculation Guidelines
    • Total enzyme volume must be 10% of total reaction volume
    • Final buffer volume must be 10% total reaction volume
    • 1000 ng DNA requires 1 uL of each enzyme
    • Ideal total volume is around 10 – 20 uL
  3. Determine volume of DNA added with required amount and DNA concentration
  4. Addition of ddH2O to make up reaction to desired volume
  5. A negative control is made with no restriction enzyme to test for any unexpected cleaving and contamination in the reagents used
Inoculating Liquid Culture
  • Asceptically pipette 5mL sterile liquid broth into a test tube. If there are no large pipette tips able to transfer 5mL of liquid broth, use a fresh falcon tube to measure.
  • Add appropriate antibiotic, if required, to desired concentration using a pipette. You will have to tilt the test tube so that the broth is close enough to the top of the tube that you can add the antibiotic to the broth. Dropping it in won’t work because you will typically be adding ~2.5mL and the drop will be too small to fall.
  • It is recommended that you make a “mastermix” with broth and antibiotics in it when you have many tubes that require the same antibiotic.