Alm:Protoplasting

Materials for 1 protoplasting run

• W1: Tris-HCl buffer, pH 8.0, 0.01M, washing solution 1, 3x wash volume so ~30ml
• R1: Tris-HCl + 0.5M sucrose, 30ml
• EDTA, 0.1M, 3.3ml
• SMM = sucrose, 0.5M , , 30 + 3x wash volume, so ~60ml
  • W2: SMM for washing, 3x wash volume so ~30ml
  • R2: SMM + lysozyme 1 mg/ml, 30ml

• R3: TSB + 0.5M sucrose, resuspension and dilution for plating, also need some for plating control with original cells 50 ml?
• TSB + 0.5M sucrose + 0.8% agarose

• 50 ml Falcon tube, 1 per protoplast type
• 25 ml serological pipets
• P1000
• P20
• plates

Regeneration:

soft agar (0.8%) plates containing

In some cases, protoplasts were resus- pended in LB medium containing 0.5 M sucrose and spread onto soft agar plates containing LB medium and 0.5 M sucrose with autoclaved plastic spreaders. The plates were subsequently incubated at either 25 or 37 1C.

In other cases, the protoplasts were diluted into molten soft agar at 45 1C containing either LB medium and 0.5 M sucrose or M9 medium, 0.5 M sucrose, and either Leu (100 mg/ml) or Arg (100 mg/ml), depending on the nutritional requirements of the auxotrophic strains used, and then poured onto soft agar plates of the same composition.

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

• supply 1 (i.e. tubes of a certain size? spreaders?)
• reagent 1
• X μL reagent 2
  • component A (reagent 2 is made up of multiple components)
  • component B
• equipment 1
• equipment 2

Procedure

Strip outer membrane

1. Pellet cells
2. Pour off supernatant
3. Resuspend in Tris-HCl
4. Add EDTA slowly
5. Shake
6. Pellet and rinse

Permeabilize cell wall

1. Pellet, rinse with SMM, resuspend in SMM + lysozyme
2. Shake and incubate

• No current step to stop lysozyme
• Should have protoplasts now

Optional: Fuse protoplasts

1. Mix aliquots of protoplasts of each parent strain
2. Add PEG
3. Step 3

Regenerate protoplasts

1. Step 1
2. Step 2
   • Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
3. Step 3
   1. Step 3 has multiple sub-steps within it.
   2. Enumerate each of those.

Notes

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

2. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
3. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
4. ISBN:0879697164

Contact

• Sean Clarke

or instead, discuss this protocol.