Streptococcus Transformation: Difference between revisions
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= | = Transformation Protocol with CTM media = | ||
* CTM | * Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees. | ||
* CTM pH 7.8 | * Inoculate fresh colonies in the 3ml of CTM pH 6.8. Vortex cultures to break up colonies. | ||
* CSP-1 peptide (in the -80 freezer; 20ul aliquots of 100uM | ** 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. | |||
== Complete Transformation Medium == | * 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. | |||
** Incubate plates in the 37°C, 5% CO2 incubator. | |||
*** Visible colonies will form after 12 hours. Plates can grow for up to 48 hours. If these strains contain a Janus cassette, using these cultures earlier is better. This will lower odds of mutations occurring in the rpsL or sacB genes, which are responsible for streptomycin and sucrose resistance/sensitivity. | |||
*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. | |||
{| class="wikitable" style="margin:auto" | |||
|+ Specifics, by transformation media | |||
|- | |||
! 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 == | |||
[[File:C+Y_SpeumoCompetence.png]] | |||
== Complete Transformation Medium (CTM) == | |||
* 3g Tryptic Soy Broth | * 3g Tryptic Soy Broth | ||
* 0.1g yeast extract | * 0.1g yeast extract | ||
Line 10: | Line 66: | ||
* 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) | * 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) | * 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 0.7 - 2.5 ug/ml (mirroring the first set of DNA), and vortex. | * 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. |
Latest revision as of 16:45, 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 cultures 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.
- Incubate plates in the 37°C, 5% CO2 incubator.
- Visible colonies will form after 12 hours. Plates can grow for up to 48 hours. If these strains contain a Janus cassette, using these cultures earlier is better. This will lower odds of mutations occurring in the rpsL or sacB genes, which are responsible for streptomycin and sucrose resistance/sensitivity.
- 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.