Contents
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
- 10 mL of overnight culture was pelleted at 5000 rpm for 5 minutes
- Pellets were resuspended in 1 mL of resuspension buffer.
- 9 mL of lysis buffer was added to resuspended pellet.
- Cell suspension was incubated at 37 C for 1 hour, shaking at 260 rpm.
- Crude cell lysate was extracted with an equal volume of phenol chloroform.
- 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.
- Crude lysate was extracted with equal volume of phenolchloroform, aqueous phase removed to a new tube.
- DNA was precipitated using ethanol precipitation. (see Other Protocols)
- DNA was dissolved in 1.5 mL nuclease free water
II. PCR Amplification of BAA-1870, BAA-1875, and WT TcdC
Protocol
- Primers were diluted to 10 uM
- dNTP was diluted to 10 mM
- DNA samples were diluted to 125 ng/uL
- The following master mix solution was prepared