Streptococcus Transformation: Difference between revisions
Jump to navigation
Jump to search
BuzzBzBuzz (talk | contribs) No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
= Transformation Protocol with CTM media = | = Transformation Protocol with CTM media = | ||
* Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees. | * Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees. | ||
* Inoculate fresh colonies | * Inoculate fresh colonies in the 3ml of CTM pH 6.8. Vortex ultures to break up colonies. | ||
** Colonies should not be more than 1-2 days old | |||
** The more colonies you inoculate, the faster the media will be ready for step 2. | |||
* Preheat a 96-well plate with 270ul CTM pH 7.8 using the 37 degree incubator with ambient CO2. | * Preheat a 96-well plate with 270ul CTM pH 7.8 using the 37 degree incubator with ambient CO2. | ||
* | * Grow cells to an Absorbance of >0.15.Make sure to vortex before checking Absorbance. When the cells have grown enough, dilute to 0.05 Absorbance. | ||
* Add the following to the 96-well plate: | |||
** Add 2ul CSP-1 peptide (more than 100 ng/ml eventual final concentration. In aliquots in the -80 freezer; 20ul aliquots of 100uM. Talk to Eric if you are not working with a D39 derivative). | ** Add 2ul CSP-1 peptide (more than 100 ng/ml eventual final concentration. In aliquots in the -80 freezer; 20ul aliquots of 100uM. Talk to Eric if you are not working with a D39 derivative). | ||
** Add DNA to 1 ug/ml final concentration — so 300ng. | ** Add DNA to 1 ug/ml final concentration — so 300ng. | ||
** If working with Janus Cassette, add 15uL. | |||
** Add 30ul of grown cells (a 1:10 dilution). | ** Add 30ul of grown cells (a 1:10 dilution). | ||
** Pipet up and down to mix; incubate for 60 minutes in the 37 degrees 5% CO2 incubator. | ** Pipet up and down to mix; incubate for 60 minutes in the 37 degrees 5% CO2 incubator. | ||
* Warm up 2.7ml TSB in a test tube for each transformation using the hot block. | * Warm up 2.7ml TSB in a test tube for each transformation using the hot block. | ||
** Must be TSB. Allows for cultures to acclimate to TSA when plated. | |||
* At the end of the 60 minute incubation, add all 300ul of the transformation to each TSB test tube. | * At the end of the 60 minute incubation, add all 300ul of the transformation to each TSB test tube. | ||
* Incubate for 60 minutes | * Incubate for 60 minutes in the 37 degrees 5% CO2 incubator. | ||
** Also works if only incubated for 30 minutes. Expression of antibiotic resistance proteins/markers occurs during this step. | |||
* Either: | * Either: | ||
## Pre-warm blood plates with appropriate antibiotics OR on a TSA plate with appropriate antibiotics. Label plates. | ## Pre-warm blood plates with appropriate antibiotics OR on a TSA plate with appropriate antibiotics. Label plates. | ||
## Add 6-7 glass beads | ## Add 6-7 glass beads | ||
### Add beads first then plate cultures so that cultures don’t splash everywhere if you throw beads onto a plate with liquid. | |||
## Plate 500ul of sample AND 25ul added catalase (if using a TSA plate), put onto the plate the same time as the bacteria | ## Plate 500ul of sample AND 25ul added catalase (if using a TSA plate), put onto the plate the same time as the bacteria | ||
## Use beads to distribute. | ## Use beads to distribute. |
Revision as of 16:33, 4 June 2024
Transformation Protocol with CTM media
- Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees.
- Inoculate fresh colonies in the 3ml of CTM pH 6.8. Vortex ultures to break up colonies.
- Colonies should not be more than 1-2 days old
- The more colonies you inoculate, the faster the media will be ready for step 2.
- Preheat a 96-well plate with 270ul CTM pH 7.8 using the 37 degree incubator with ambient CO2.
- Grow cells to an Absorbance of >0.15.Make sure to vortex before checking Absorbance. When the cells have grown enough, dilute to 0.05 Absorbance.
- Add the following to the 96-well plate:
- Add 2ul CSP-1 peptide (more than 100 ng/ml eventual final concentration. In aliquots in the -80 freezer; 20ul aliquots of 100uM. Talk to Eric if you are not working with a D39 derivative).
- Add DNA to 1 ug/ml final concentration — so 300ng.
- If working with Janus Cassette, add 15uL.
- Add 30ul of grown cells (a 1:10 dilution).
- Pipet up and down to mix; incubate for 60 minutes in the 37 degrees 5% CO2 incubator.
- Warm up 2.7ml TSB in a test tube for each transformation using the hot block.
- Must be TSB. Allows for cultures to acclimate to TSA when plated.
- At the end of the 60 minute incubation, add all 300ul of the transformation to each TSB test tube.
- Incubate for 60 minutes in the 37 degrees 5% CO2 incubator.
- Also works if only incubated for 30 minutes. Expression of antibiotic resistance proteins/markers occurs during this step.
- Either:
- Pre-warm blood plates with appropriate antibiotics OR on a TSA plate with appropriate antibiotics. Label plates.
- Add 6-7 glass beads
- Add beads first then plate cultures so that cultures don’t splash everywhere if you throw beads onto a plate with liquid.
- Plate 500ul of sample AND 25ul added catalase (if using a TSA plate), put onto the plate the same time as the bacteria
- Use beads to distribute.
- Allow plates to completely dry in hood.
- Or,
- 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 TSB soft agar 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 with a second plate, 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 for 1-2 days in 37 degree, 5% CO2 incubator
For transformation not using CTM media, use the following media combinations below.
Media 'A' (Growth Media) | Media 'B' (Transformation Media) | Grow to Absorbance |
---|---|---|
C+Y pH 7.4 | C+Y pH 7.4 | 0.52 Absorbance |
CTM pH 6.8 | CTM pH 7.8 | 0.39 Absorbance |
BHI pH 7.4 | 430ul culture + 50ul 100mM NaOH + 5ul 20% BSA + 2ul 100mM CaCl2 | 0.1 Absorbance |
Columbia pH 7.4 | Columbia pH 6.6 | 0.05 Absorbance. With our Spectrometer, impossible. Use 0.13 Absorbance; dilute 4-fold with pre-warmed Columbia pH 7.4; wait 30 minutes and then use. |
Recipes
C+Y Recipe
Complete Transformation Medium (CTM)
- 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)
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.