Streptococcus Transformation: Difference between revisions
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PreDec2022>EricMiller No edit summary |
PreDec2022>EricMiller No edit summary |
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== Reagents Needed== | == Reagents Needed== | ||
* CTM (Complete Transformation Medium) pH | * CTM (Complete Transformation Medium) pH 6.8 | ||
* CTM pH 7. | * CTM pH 7.8 | ||
* 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. | * 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 == | == Complete Transformation Medium == | ||
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# Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance | # 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. | # 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 100 ng/ml eventual final concentration. | # 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 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). | # Add 30ul of grown cells (a 1:10 dilution). | ||
Revision as of 22:52, 23 September 2021
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 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
- 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.