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- 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 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).
- Incubate at 37 degrees for 60 minutes using the hot block.
- During this time, warm up 2.7ml CTM pH 6.8 in a test tube for each transformation using the hot block.
- At the end of the 60 minute incubation, add all 300ul of the transformation to each test tube. Be sure to label which test tube is which.
- Incubate for 60 minutes in the 37 degree incubator.
- During this second 60 minute incubation, change the hot block to 42 degrees and put in 2 empty test tubes per transformation.
- Melt and pipet 3.5ml soft agar (either TSB or HTY) into each test tube.
- Warm up TSB plates with appropriate antibiotics by placing them in either 37 degree incubators. Move to the hood and label plates.
- After the second 60 minute incubation, add 10ul of catalase and 10ul of TCC to each soft agar tube. There's no need to add antibiotics to the soft agar.
- Add 2 x 750ul of the transformation to a soft agar tube (using a micropipettor, not a serological pipet), immediately vortex for 3-5 seconds and immediately pour onto a TSB plate. The agar will solidify quickly, so do this step one soft agar tube at a time.
- Repeat the above step, so that all 3ml of the transformation is used. For (-), no DNA controls, feel free to only plate a total of 1.5ml onto a single TSB plate. Repeat for all transformations, one soft-agar tube at a time.
- Wait 3-5 minutes for all plates to solidify
- Incubate the plate overnight in the 37 degrees C 5% CO2 incubator.
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.