Team:Mississippi State/Recipes

From 2008.igem.org

Contents

1% Agarose Gel for Electrophoresis

  1. WEAR GLOVES
  2. Needed: 250ml flask, Agarose, 1xTAE Buffer, ddH2O in squirt bottle, Balance, Spatula, Balance tray, Ethydium Bromide.
  3. Weigh out 0.25g Agarose, adding to flask.
  4. Add 25ml 1xTAE Buffer.
  5. Weigh flask and contents, remembering weight.
  6. Swirl until dissolved.
  7. Microwave until boil 2-3 times.
  8. Allow to cool until just cool enough to touch.
  9. Reweigh flask and contents, adding ddH2O until original weight.
  10. Add 5ul EtBr, swirl.
  11. Pour flask contents into Gel tray, allow to solidify.

Glycerol Stock for Long-Term Storage

  1. Streak original colony out on a Low Salt LB plate containing 100ug/ml blasticidin. Incubate plate at 37C overnight.
  2. isolate single colony and inoculate into 1-2ml of Low Salt LB containing 100ug/ml blasticidin.
  3. Grow the culture to mid-log phase (OD600 = 0.5=0.7).
  4. Mix 0.85ml of culture with 0.15ml sterile glycerol and transfer to a cryovial.
  5. Store at -80C.

Low Salt LB Medium with Blasticidin

  • 10g Tryptone
  • 5g NaCl
  • 5g Yeast Extract
  1. 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.
  2. Autoclave on liquid cycle at 15psi and 121C for 20min.
  3. Allow the medium to cool to at least 55C before adding the blasticidin to 100ug/ml final concentration.
  4. Store plates at +4C in dark. Plates containing blasticidin are stable for up to 2 weeks.

Pfx50 PCR Mix

  • Template, Primer(s), Pfx50 Polymerase, Pfx50 Buffer, dNTP, ddH2O, PCR Tube, vortex, micro centrifuge
  • Add contents as follows to PCR Tube for 50ul total:


Taq PCR Mix

  • Template, Primer(s), Taq Polymerase, 10xTaq Buffer, MgCl2, dNTP, ddH2O, PCR Tube, vortex, micro centrifuge
  • Add contents as follows to PCR Tube for 50ul total:
  1. 39ul ddH2O
  2. 5ul 10xTaq Buffer
  3. 1ul dNTP mix
  4. 1.5ul MgCl2
  5. 2ul(total) Primer(s)
  6. 1ul Template
  7. 0.5ul Taq Polymerase
  8. Label Tube, vortex, and spin.
  9. Ready for PCR.

YPD (+Blasticidin) Yeast Extract Peptone Dextrose Medium (1 liter)

  • 1% yeast extract
  • 2% peptone
  • 2% dextrose (glucose)
  • +/-2% agar
  • +/- appropriate concentration of blasticidin
  1. Dissolve: 10g yeast extract 20g peptone in 900ml water.
  2. Include 20g of agar if making YPD slants or plates.
  3. Autoclave for 20min on liquid cycle.
  4. Add 100ml of 20% dextrose (filter-sterilize dextrose before use).
  5. 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.
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YPDS + Blasticidin Agar

  • 1% yeast extract
  • 2% peptone
  • 2% dextrose (glucose)
  • 1M sorbitol
  • +/-2% agar
  • +/- appropriate concentration of blasticidin
  1. Dissolve: 10g yeast extract 182.2g sorbitol 20g peptone in 900ml water.
  2. Include 20g of agar if making YPD slants or plates.
  3. Autoclave for 20min on liquid cycle.
  4. Add 100ml of 20% dextrose (filter-sterilize dextrose before use).
  5. 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.
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