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Team:Mississippi State/Recipes
From 2008.igem.org
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(→Low Salt LB Medium with Blasticidin) |
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#Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies. | #Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies. | ||
*Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks. | *Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks. | ||
| - | *[[Team:Mississippi State|Home]] | + | *[[Team:Mississippi State/Notebook|Home]] |
==YPDS + Blasticidin Agar== | ==YPDS + Blasticidin Agar== | ||
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#Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies. | #Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies. | ||
*Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks. | *Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks. | ||
| - | *[[Team:Mississippi State|Home]] | + | *[[Team:Mississippi State/Notebook|Home]] |
==Glycerol Stock for Long-Term Storage== | ==Glycerol Stock for Long-Term Storage== | ||
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#Mix 0.85ml of culture with 0.15ml sterile glycerol and transfer to a cryovial. | #Mix 0.85ml of culture with 0.15ml sterile glycerol and transfer to a cryovial. | ||
#Store at -80C. | #Store at -80C. | ||
| - | *[[Team:Mississippi State|Home]] | + | *[[Team:Mississippi State/Notebook|Home]] |
Revision as of 16:18, 16 June 2008
Contents |
Low Salt LB Medium with Blasticidin
- 10g Tryptone
- 5g NaCl
- 5g Yeast Extract
- Combine dry reagents above and add deionized, distilled water to 950ml. Adjust pH to 7.0 with 1N NaOH. Bring the volume up to liter. For plates, add 15g/L agar before autoclaving.
- Autoclave on liquid cycle at 15psi and 121C for 20min.
- Allow the medium to cool to at least 55C before adding the blasticidin to 100ug/ml final concentration.
- Store plates at +4C in dark. Plates containing blasticidin are stable for up to 2 weeks.
YPD (+Blasticidin) Yeast Extract Peptone Dextrose Medium (1 liter)
- 1% yeast extract
- 2% peptone
- 2% dextrose (glucose)
- +/-2% agar
- +/- appropriate concentration of blasticidin
- Dissolve: 10g yeast extract 20g peptone in 900ml water.
- Include 20g of agar if making YPD slants or plates.
- Autoclave for 20min on liquid cycle.
- Add 100ml of 20% dextrose (filter-sterilize dextrose before use).
- Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies.
- Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks.
- Home
YPDS + Blasticidin Agar
- 1% yeast extract
- 2% peptone
- 2% dextrose (glucose)
- 1M sorbitol
- +/-2% agar
- +/- appropriate concentration of blasticidin
- Dissolve: 10g yeast extract 182.2g sorbitol 20g peptone in 900ml water.
- Include 20g of agar if making YPD slants or plates.
- Autoclave for 20min on liquid cycle.
- Add 100ml of 20% dextrose (filter-sterilize dextrose before use).
- Cool solution to ~60C and add the appropriate amount of blasticidin from a 10mg/ml stock solution. Note: It is necesary to include blasticidin in the medium for selection of Pichia transformants only. Blasticidin may be omitted from the medium when performing expression studies.
- Store YPD slants or plates containing blasticidin at +4C. The shelf life is one to two weeks.
- Home
Glycerol Stock for Long-Term Storage
- Streak original colony out on a Low Salt LB plate containing 100ug/ml blasticidin. Incubate plate at 37C overnight.
- isolate single colony and inoculate into 1-2ml of Low Salt LB containing 100ug/ml blasticidin.
- Grow the culture to mid-log phase (OD600 = 0.5=0.7).
- Mix 0.85ml of culture with 0.15ml sterile glycerol and transfer to a cryovial.
- Store at -80C.
