Biomolecular Breadboards:Protocols:3-PGA Buffer Prep

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Preparation of 3-PGA Buffer for the Cell-Free Expression System, 14X

Component Stock Final Concentration in cell-free reaction 1-fold 4-fold 6-fold
HEPES pH8 2M 50mM 150µl 600µl 900µl
Water 6µl 24µl 36µl
Homemade Nucleotides 156mM: ATP & GTP 94mM: CTP & UTP 1.5mM: ATP & GTP 0.9mM: CTP & UTP 57.8µl 231.2µl 346.8µl
tRNA E.coli 50mg/ml 0.2mg/ml 24µl 96µl 144µl
CoA 65mM 0.26mM 24µl 96µl 144µl
NAD 175mM 0.33mM 11.5µl 46µl 69µl
cAMP 650mM 0.75mM 7.1µl 28.4µl 42.6µl
Folinic Acid 33.9mM 0.068mM 12µl 48µl 72µl
Spermidine 1M 1mM 6µl 24µl 36µl
3-PGA 1.4M 30mM 128.6µl 514.4µl 771.6µl
Total volume 427µl 1708µl 2562µl
Aliquot volume 7µl 7µl 7µl
Tube number 60 240 360

Each component can be prepared in advance and stored in -80°C: this step might be even better than fresh preparation because of keeping same concentration.

  • Take components out at room temperature from -40°C or -20°C (tRNA) 30min before weighting them in order to avoid condensation.
  • Take spermidine out from 4°C and place it near heat block to slightly warm the vial (melting).
  • Keep spatula clean all the time: rinse with water and isopropanol, then dry with air gun.
    • Good aliquot volume of each component is for 4-folds preparation.
  • Weigh each component in a micro-centrifuge tube one by one and dissolve it under its own protocol below.
  • Store all components in -80°C.
  • Once a few tens of 3-PGA buffer tubes are left, prepare new 3-PGA buffer with components which are already prepared individually and stored in -80°C.

First, prepare homemade nucleotides:

  • ATP + GTP + UTP + CTP, then add KOH.
  • Each time, slowly mix components.

Add component in the order of the table above from top to bottom: mix HEPES and water, and then add nucleotides and tRNA and so on.

Whenever adding one component, vortex it to mix.

Preparation of each component below is for stock preparation and aliquot volume is for 4-folds.

Hepes KOH pH8

  • Supplier: sigma, appearance: solid.
  • Product Number: H6147-100G
  • MW=238.31
  • Adjust with KOH to pH8
  • 476.62mg/ml in water = 2M
  • Large stock is on the shelf

Homemade Nucleotides

  • ATP dipotassium salt dihydrate(sigma, A8937-1G, appearance: solid): MW=619.4 → prepare 540mM (in water)
    • Dissolve 500mg of ATP in 1495µl of water in total volume: 500mg is counted as 250µl, therefore add 1245µl of water.
    • Aliquot it by 77µl
  • GTP disodium salt (USB, 16800, appearance: solid): MW=567.14 → prepare 760mM (in water)
    • Dissolve 500mg of GTP in 1160µl of water in total volume: 500mg is counted as 250µl and add 910µl water.
    • Aliquot it by 56µl
  • UTP trisodium salt dihydrate (USB, 23160, appearance: solid): MW=586.12 → prepare 813mM (in water)
    • Dissolve 300mg of UTP in 630µl of water in total volume: 300mg is counted as 150µl and add 480µl water.
    • Aliquot it by 33µl
  • CTP disodium salt dihydrate (USB, 14121, appearance: solid): MW=563.16 → prepare 621mM (in water)
    • Dissolve 300mg of CTP in 858µl of water in total volume: 300mg is counted as 150µl and add 708µl water.
    • Aliquot it by 42µl
  • 74µl ATP at 540mM + 52.8µl GTP at 760mM + 29.6µl UTP at 813mM + 38.8µl CTP at 621mM = 195.2µl
  • Add 60µl of KOH at 15% (dilute 3 times in water from 45% stock (sigma, 417661)) to get pH at 7.5 → therefore total volume of homemade nucleotides is 255.2µl

tRNA at 50 mg/ml in water

  • E.coli total tRNA from MRE600 (appearance: solid)
  • Supplier: Roche
  • Product Number: 109541 (ustores); 10109541001 for cat #
  • Prepare 50mg/ml → Dissolve 50mg of tRNA in water in a total volume of 1 ml final.
  • Aliquot by 100µl

CoA at 65 mM in water

  • Coenzyme A hydrate yeast (appearance: solid)
  • Supplier: sigma
  • Product Number: C4282
  • MW=767.53
  • Prepare 65mM (50mg/ml) → Dissolve 50mg of CoA in water in a total volume of 1 ml final.
  • Aliquot it by 100µl

NAD at 175 mM

  • Beta-nicotinamide adenine dinucleotide hydrate (appearance: solid)
  • Supplier: sigma
  • Product Number: N6522
  • MW=663.43
  • Adjust pH with Tris 2M to get 7.5~8
  • Prepare 175mM → 100mg of NAD +727µl of water +77µl of Tris at 2M
  • Aliquot by 50µl

cAMP at 650 mM

  • Adenosine 3’,5’ cyclic monophosphate (appearance: solid)
  • Supplier: sigma
  • Product Number: A9501
  • MW=329.22
  • Adjust with Tris 2M to pH8
  • Prepare 650mM → 150mg of cAMP + 348µl of water + 257µl of Tris at 2M
  • Aliquot it by 32µl

Folinic acid at 33.9 mM

  • Folinic acid calcium salt (appearance: solid)
  • Supplier: sigma
  • Product Number: F7878
  • MW=511.50
  • Prepare 33.9mM → 20mg + 1.1534ml of water (solid volume is negligible)
  • Slightly green color
  • Aliquot by 51µl

Spermidine at 1M in water

  • Supplier: sigma (pure component, mix solid/liquid, melt at RT)
  • Product Number: 85558
  • MW=145.25
  • Liquid, store at 4°C
  • Density at 25℃: 0.925g/ml = 6.37M
  • Prepare 1M → 100µl at 6.37M + 537µl of water ← 637µl total volume
  • Aliquot by 27µl

3-PGA at 1.4 M

  • D-(-)-3-phosphoglyceric acid disodium salt (appearance: solid)
  • Supplier: sigma
  • Product number: P8877
  • MW=230.02
  • Adjust with Tris 2M to pH7.5
  • 3g + 3.156ml water + 5.04ml of Tris at 2M = 1.4M
  • Aliquot by 520µl