Biomolecular Breadboards:Protocols:Amino Acids Prep

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Amino Acid Preparation for the Cell-Free Expression System

Information:

• RTS Amino Acid Sampler: 5 PRIME, product number 2401530.
• Store at -20°C.
• Do not order many boxes at once because the expiration date is not long.
• 1 kit = 1600 aliquots of 26µl (volume required for a cell-free reaction of 90µl total).

Preparation for 1 full kit:

• It is better to prepare an entire kit (although half of the kit can be also prepared).
• The stock of each individual amino acid is at 168mM except Leucine at 140mM.
• Total volume of each individual amino acid is at 1.5ml.
• Once all components are mixed, the final concentration of each amino acid is at 6mM except Leucine at 5mM.

• Amino acids hard to dissolve: Asn and Phe, and even harder: Cys
  • Take them out from -20°C one hour before all others
  • Incubate on bench at RT until melting.
  • Once melted, place in the incubator at 37°C.
  • Frequently, vortex them and put them back to 37°C
  • Do not put Phe on the ice even when completely dissolved!
• Take all others out and place them on bench or rotator one hour after AsN, Phe and Cys.
• Vortex each amino acid tube from time to time.
• Once every component is dissolved (except that Cys wouldn’t dissolve, but when all others are dissolved, it is time to go), start to mix one by one.

• To avoid precipitation, add Trp, Tyr, Leu and then Cys at the very end.
  • Use 50ml falcon: total volume would be 42ml.
  • Add 12ml of water into it.
  • Then add 1.5ml of each amino acid.
  • Whenever one component is added, vortex them to mix well.
  • Add Cys at the end. Cys is still not dissolved well, so once added, warm up little bit with your hands until it is dissolved completely (clear transparency).
• Aliquot:
  • For one reaction: 26µl
  • For one feeding (basically 10-folds): 230µl
  • Use liquid nitrogen to freeze amino acids quickly, then transfer them to -80°C