Streptococcus Transformation

Reagents Needed

• CTM (Complete Transformation Medium) pH 7.1
• CTM pH 7.9
• CSP-1 peptide (in the -80 freezer) or CSP-2 peptide (which we do not have). Talk to Eric if you are not working with a D39 derivative.

Complete Transformation Medium

• 3g Trypic Soy Broth
• 0.1g yeast extract
• Fill up to 100ml water and autoclave
• Add to a final concentration filter sterilized 1mM CaCl2 and filter sterilized 0.2% BSA (Bovine Serum Albumin)

Competence Protocol

1. Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
2. Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance
3. Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 37 degrees using the hot block.
4. Add CSP-1 peptide to this tube to 100 ng/ml eventual final concentration. This is then adding 30ng of CSP-1, as our final volume will eventually be 300ul.
5. Add DNA
6. Add 30ul of grown cells (a 1:10 dilution).
7. Vortex
8. Incubate at 37 degrees using the hot block for 60 minutes.
9. Plate cells on a blood TSA plate that has appropriate antibiotics in it.

Competence Protocol with CRISPR

• As above, but add the editing construct at a final concentration of 2.5ug/ul
• Incubate at 37 degrees using the hot block for 20 minutes.
• Add the CRISPR targeting construct at a final concentration of 2.5ug/ul, and vortex.
• Incubate at 37 degrees using the hot block for 40 minutes.