Streptococcus Transformation

Reagents Needed

CTM (Complete Transformation Medium) pH 6.8
CTM pH 7.8
CSP-1 peptide (in the -80 freezer; 20ul aliquots of 100uM) or CSP-2 peptide (which we do not have). Talk to Eric if you are not working with a D39 derivative.

3g Tryptic Soy Broth
0.1g yeast extract
Fill up to 100ml MilliQ water and autoclave
Add to a final concentration filter sterilized 1mM CaCl2 (found on chemical shelf), filter sterilized 0.2% BSA (Bovine Serum Albumin), and filter sterilized 1X trace mineral solution (found on chemical shelf)

Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance
Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 37 degrees using the hot block.
Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
Add DNA to 1 ug/ml final concentration — so 300ng. If this is too much DNA, it might work with half of the amount — 150ng of DNA.
Add 30ul of grown cells (a 1:10 dilution).
Vortex
Incubate at 37 degrees using the hot block for 60 minutes.
Plate cells on a blood TSA plate that has appropriate antibiotics in it. Use sterile cotton swab to spread the cell mixture.
Incubate the plate overnight in the 37 degrees C 5% CO2 incubator.

As above, but add the editing construct at a final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA)
Incubate at 37 degrees using the hot block for 20 minutes.
Add the CRISPR targeting construct at a final concentration of 0.7 - 2.5 ug/ml (mirroring the first set of DNA), and vortex.
Incubate at 37 degrees using the hot block for 40 minutes.