Team:McMasterU/CDifficile

Compiled Protocols for TcdC Plasmids Design



I. C. Difficile gDNA Extraction Using Phenolchloroform


Materials


Resuspension Buffer

    10 mM Tris-HCl, pH 8.0
    20% (w/v) Sucrose

Lysis Buffer

    10 mM Tris-HCl, pH 8.0
    1mM EDTA
    1% (w/v) SDS

RNase A (10mg/mL)

    10 mg bovine pancreating RNase A
    10 mM Tris, pH 8.0
    500 mM NaCl
    850 uL water
    Heated to 95 C until use



Protocol

  1. 10 mL of overnight culture was pelleted at 5000 rpm for 5 minutes
  2. Pellets were resuspended in 1 mL of resuspension buffer.
  3. 9 mL of lysis buffer was added to resuspended pellet.
  4. Cell suspension was incubated at 37 C for 1 hour, shaking at 260 rpm.
  5. Crude cell lysate was extracted with an equal volume of phenol chloroform.
  6. Aqueous phase was recovered to a new tube and 25 ug RNase A was added, crude lysate was incubated at 37 C for 20 minutes.
  7. Crude lysate was extracted with equal volume of phenolchloroform, aqueous phase removed to a new tube.
  8. DNA was precipitated using ethanol precipitation. (see Other Protocols)
  9. DNA was dissolved in 1.5 mL nuclease free water



II. PCR Amplification of BAA-1870, BAA-1875, and WT TcdC


Protocol

  1. Primers were diluted to 10 uM
  2. dNTP was diluted to 10 mM
  3. DNA samples were diluted to 125 ng/uL
  4. The following master mix solution was prepared