Streptococcus Transformation: Difference between revisions
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PreDec2022>EricMiller No edit summary |
PreDec2022>Dcapcha No edit summary |
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== Complete Transformation Medium == | == Complete Transformation Medium == | ||
* 3g | * 3g Tryptic Soy Broth | ||
* 0.1g yeast extract | * 0.1g yeast extract | ||
* Fill up to 100ml water and autoclave | * Fill up to 100ml water and autoclave | ||
* Add to a final concentration filter sterilized 1mM CaCl2 | * Add to a final concentration filter sterilized 1mM CaCl2, filter sterilized 0.2% BSA (Bovine Serum Albumin), and trace mineral solution (1X) | ||
== Competence Protocol == | == Competence Protocol == |
Revision as of 16:24, 17 June 2022
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.
Complete Transformation Medium
- 3g Tryptic Soy Broth
- 0.1g yeast extract
- Fill up to 100ml water and autoclave
- Add to a final concentration filter sterilized 1mM CaCl2, filter sterilized 0.2% BSA (Bovine Serum Albumin), and trace mineral solution (1X)
Competence Protocol
- 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.
Competence Protocol with CRISPR
- 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.