Jul 22, 2022

Public workspacerNTPs Stock Preparation / IVT Standard Reaction

DOI
dx.doi.org/10.17504/protocols.io.e6nvwkpx2vmk/v1
  • 1Universidad de Chile;
  • 2Pontificia Universidad Catolica de Chile
Felipe Navarro Martínez
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwkpx2vmk/v1
Protocol Citation: Felipe Navarro Martínez, Anibal Arce Medina, Fernan Federici 2022. rNTPs Stock Preparation / IVT Standard Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkpx2vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 12, 2021
Last Modified: July 22, 2022
Protocol Integer ID: 55862
Abstract
This protocol describes the preparation of rNTPs stock solutions for their use on IVT and other molecular reactions.

The rNTPs preparation is adapted from a protocol by Berglund Lab (Goers, 2006), it contains instructions to prepare Concentration100 millimolar (mM) stock solutions of each ribonucleotide with a final pH of 7-8 each. It is important to consider that each rNTP has a slightly different molar mass, and this value can also vary according to the degree of hydration of the reagent and provider. The molar mass value you should use to determine the resuspension volume must be the one indicated on the flask that contains each rNTP.

In our case, we have:
ATP (583,36 g/mol)
GTP (567,10 g/mol)
UTP (550,09 g/mol)
CTP (527,12 g/mol)

The second part of this protocol describes the preparation of an in vitro transcription (IVT) reaction of fluorescent RNA aptamers, like Spinach or Broccoli. In our lab, we use it to test, validate and standardize our buffers, enzymes, ribonucleotides and other reagents. Spinach and Broccoli aptamers bind to DFHBI-1T and this interaction “mimics a GFP fluorophore”, as the reaction product emits a green fluorescence that can be observed in a UV transilluminator.

The homemade IVT-aptamer reaction was based on procedures used by the Adamala-Engelhart Laboratory (Heili et al., 2018 & Aufdembrink et al., 2020), with our own modifications.

References
- Aufdembrink, L. M., Khan, P., Gaut, N. J., Adamala, K. P., & Engelhart, A. E. (2020). Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout. Rna, 26(9), 1283-1290.
- Goers, E. (2006). Berglund: Making rNTP - OpenWetWare. OpenWetWare. https://openwetware.org/wiki/Berglund:_making_rNTP
- Heili, J. M., Gomez-Garcia, J., Gaut, N. J., Cash, B. W., Aufdembrink, L. M., Heffron, B. A., ... & Engelhart, A. E. (2018). Real-time visualization of in vitro transcription of a fluorescent RNA aptamer: an experiment for the upper-division undergraduate or first-year graduate laboratory.Journal of Chemical Education,95(10), 1867-1871.
- Paige, J. S., Wu, K. Y., & Jaffrey, S. R. (2011). RNA mimics of green fluorescent protein. Science (New York, N.Y.), 333(6042), 642–646.

Materials
For rNTPs stock
  • NTPs
  • NaOH Concentration1 Molarity (M) Solution
  • 1:1 HCl Solution
  • Nuclease-Free Water
  • pH Strips

For IVT Standard Reaction
  • 5X In Vitro Transcription Buffer
  • rNTPs, 100 mM each
  • T7 Polymerase (2 mg/mL)
  • DMSO
  • Nuclease-Free Water
  • DFHBI-1T 40 mM

Protocol materials
ReagentDFHBI-1TMerck MilliporeSigma (Sigma-Aldrich)Catalog #SML2697-25MG
Step 10
rNTPs Preparation
rNTPs Preparation
Determine the mass of rNTPs that is going to be used. In this case, Amount100 mg of each rNTP are recommended.

Calculate the necessary volume to obtain a Concentration100 millimolar (mM) solution of each rNTP.

We did this by estimating how many mmoles are in 100mg and used that value to estimate the volume for each 100mM solution. In our case, we calculated:
- ATP (583,36 g/mol) --> 1714 uL
- GTP (567,10 g/mol) --> 1763 uL
- UTP (550,09 g/mol) --> 1818 uL
- CTP (527,12 g/mol) --> 1898 uL

If you are using 100 mg of each rNTP, this result should be around Amount1600-2100 µL


Weight Amount100 mg of each rNTP separately in an 1,5 mL Eppendorf tube. Deposit them on ice.

We recommend doing this step slowly, as we experienced a lot of static between the reagents and the tube when we were doing this.


Add Amount1300 µL of nuclease-free water to each tube. Vortex and deposit them on ice.

Since you need to adjust the pH of the solution, we recommend adding less water at first than the amount you will actually need so you can have a broad range of volume available for the next step.

Measure the pH of the solution using a pH strip.
Adjust the pH of the solution using NaOH Concentration1 Molarity (M) and HCl 1:1 to range between 7-8

rNTPs solutions are originally very acidic (pH 2-5), so you will probably need to add only base. The HCl solution is only required if you overshoot the pH by adding too much NaOH. We recommend adding the first Amount200 µL of NaOH 1M and from there continue in volumes betweenAmount5-20 µL until you get to the pH 7-8 range. Measure the pH each time with a pH strip and have a record of how much volume you are adding to each solution. Be careful to not go over the final volume.

Critical
If needed, add nuclease-free water up to the final volume of each solution. Vortex each tube.
Aliquot each tube separately, label them and store them atTemperature-20 °C until use.

IVT Standard Reaction
IVT Standard Reaction
3h
3h
5X IVT buffer

Our 1X IVT buffer is composed of Tris Acetate Ph8.1 Concentration30 millimolar (mM) , Magnesium glutamate Concentration24 millimolar (mM) , Potassium glutamate Concentration90 millimolar (mM) , Spermidine Concentration2 millimolar (mM) and DTT Concentration1 millimolar (mM)
ABCD
Stock solutionsVolume for a 50mL solution5X Concentration1X concentration
Tris-Ac pH 8.1 500mM3 ml150 mM30 mM
Mg-Glutamate 500mM2.4 ml120 mM24 mM
K-Glutamate 100mM4.5 ml450 mM90 mM
Spermidine 1M100 uL10 mM2 mM
DTT 1M50 uL5 mM1 mM
Considerations should be made that these concentrations can be further optimized.
We aliquote this 5x transcription buffer and store it at Temperature-20 °C
DFHBI-1T 40 mM

In case Broccoli or Spinach aptamer are used to benchmark the quality of the IVT reaction, it is necessary to prepare the following:

For a Concentration40 millimolar (mM) solution, mix Amount5 mg of ReagentDFHBI-1TSigma AldrichCatalog #SML2697-25MG with Amount390 µL of DMSO. Vortex, aliquot in 1,5 mL Eppendorf tubes and save at Temperature-20 °C covered with aluminium foil.

Optional
Test Reaction Protocol

The amounts shown here are for a single Amount20 µL reaction.
Remember to thaw, vortex and quickly spin tubes down before opening for dispensing. It is preferable to keep all the tubes on ice when not in use. When stored, all the reagents should be at Temperature-20 °C .
ABC
1x (uL)Final concentration
IVT Buffer 5x41X
ATP 100 mM110 mM
CTP 100mM110 mM
UTP 100mM110 mM
GTP 100mM110 mM
DFHBI-1T 40 mM12 mM (if needed)
T7 Pol Mix (2 mg/mL)20,1 mg/mL
H207
DNA2Spinach or Broccoli aptamer
20
Although we have not formally tested this, we followed the general recommendations of using linear dsDNA rather than plasmids as a substrate for IVT. We normally use linear dsDNA from a cleaned up-PCR reaction at a concentration ranging from 70-200 ng/uL stock.

Incubate the reaction at Temperature37 °C for Duration03:00:00 for downstream applications, it can also be incubated overnight, however little increase in yield is seen in our hands.
Note
Although not necessary. We observed an increase in the reaction yields by supplementing the reaction with Amount1 µL of ReagentPyrophosphatase, Inorganic (E.coli) - 10 unitsNew England BiolabsCatalog #M0361S




3h