Team:Mississippi State/Recipes

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.
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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.
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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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