Streptococcus Transformation: Difference between revisions
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PreDec2022>EricMiller (Created page with "== Reagents Needed== * CTM (Complete Transformation Medium) pH 6.8, without Calcium Chloride or Bovine Serum Albumin (BSA) * CTM pH 7.8, with filtered Calcium Chloride added a...") |
PreDec2022>EricMiller No edit summary |
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== Reagents Needed== | == Reagents Needed== | ||
* CTM (Complete Transformation Medium) pH | * CTM (Complete Transformation Medium) pH 7.1 | ||
* CTM pH 7. | * 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. | * 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 == | |||
# Freshly grow up single colonies on a blood TSA plate of the strain to be transformed. | # 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 | # 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 | # 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 | # 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. | ||
# Add DNA | # Add DNA | ||
# Add 30ul of grown cells (a 1:10 dilution). | # Add 30ul of grown cells (a 1:10 dilution). | ||
# Vortex | # Vortex | ||
# Incubate at | # Incubate at 37 degrees using the hot block for 60 minutes. | ||
# Plate cells on a blood TSA plate that has appropriate antibiotics in it. | # 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. | |||
Revision as of 13:34, 8 July 2021
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
- 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 100 ng/ml eventual final concentration. This is then adding 30ng of CSP-1, as our final volume will eventually be 300ul.
- Add 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 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.