Streptococcus Transformation: Difference between revisions
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# 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. This is then adding 30ng of CSP-1, as our final volume will eventually be 300ul. | # 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 to 1 ug/ml final concentration. | # 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). | ||
# Vortex | # Vortex | ||
Line 22: | Line 22: | ||
== Competence Protocol with CRISPR == | == Competence Protocol with CRISPR == | ||
* As above, but add the editing construct at a final concentration of 2. | * 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. | * Incubate at 37 degrees using the hot block for 20 minutes. | ||
* Add the CRISPR targeting construct at a final concentration of 2. | * 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. | * Incubate at 37 degrees using the hot block for 40 minutes. |
Revision as of 13:40, 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 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.