Biomolecular Breadboards:Protocols:Amino Acids Prep: Difference between revisions

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==Amino Acid Preparation for the Cell-Free Expression System==
==Amino Acid Preparation for the Cell-Free Expression System==
'''UPDATE (4-21-15): The RTS Amino Acid Sampler is now available from biotechrabbit, which is located in and ships from Germany. See http://www.biotechrabbit.com/rts-amino-acid-sampler-399.html. The current approximate price including shipping to CA is $395.'''
Alternatively, see Vincent's new protocol at: http://www.biotechniques.com/multimedia/archive/00248/BTN_A_000114249_O_248780a.pdf


====Information:====
====Information:====
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*Once all components are mixed, the final concentration of each amino acid is at 6mM except Leucine at 5mM.
*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
'''Amino acids hard to dissolve: Asn and Phe, and even harder:  Cys'''
**Incubate on bench at RT until melting.
*Take them out from -20°C one hour before all others
**Once melted, place in the incubator at 37°C.
*Incubate on bench at RT until melting.
**Frequently, vortex them and put them back to 37°C
*Once melted, place in the incubator at 37°C.
**'''Do not put Phe on the ice even when completely dissolved!'''
*Frequently, vortex them and put them back to 37°C
*Take all others out and place them on bench or rotator one hour after AsN, Phe and Cys.
*'''Do not put Phe on the ice even when completely dissolved!'''
 
 
All other amino acids: take them out and place them on bench or rotator one hour after AsN, Phe and Cys.
*Vortex each amino acid tube from time to time.
*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.
*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.
'''To avoid precipitation, add Trp, Tyr, Leu and then Cys at the very end.'''
**Add 12ml of water into it.
*Use 50ml falcon: total volume would be 42ml.
**Then add 1.5ml of each amino acid.
*Add 12ml of water into it.
**Whenever one component is added, vortex them to mix well.
*Then add 1.5ml of each amino acid.
**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).
*Whenever one component is added, vortex them to mix well.
*Aliquot:  
*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).
**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
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

Latest revision as of 16:51, 21 April 2015

Home Protocols DNA parts Preliminary Data Models More Info


Amino Acid Preparation for the Cell-Free Expression System

UPDATE (4-21-15): The RTS Amino Acid Sampler is now available from biotechrabbit, which is located in and ships from Germany. See http://www.biotechrabbit.com/rts-amino-acid-sampler-399.html. The current approximate price including shipping to CA is $395.


Alternatively, see Vincent's new protocol at: http://www.biotechniques.com/multimedia/archive/00248/BTN_A_000114249_O_248780a.pdf

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!


All other amino acids: take them 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

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