Nov 21, 2024

Public workspaceDDO in vitro transcription

  • 1Washington University
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Protocol CitationCarolina Lopez 2024. DDO in vitro transcription. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49qprgo5/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: May 28, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 100765
Abstract
Protocol for in vitro transcription of DDO and QC
Materials


Perform all steps with RNase-free reagents in RNase-free tubes in the hood after surface decontamination with RNase Zap.
I. Plasmid Preparation
I. Plasmid Preparation
A. Linearization: in a PCR microtube
Amount5 µg of rDVG plasmid
Amount5 µL CutSmart Buffer BioLabs
Amount5 µL EcoRI-HF BioLabs
• H2O up to Amount50 µL
Incubation time: overnight in Temperature37 °C using a PCR machine.

B. Plasmid DNA Precipitation: Transfer to Amount1.5 mL RNase free tube and add:
Amount4 µL Linear acrylamide
Amount2.5 µL Concentration0.5 Molarity (M) EDTA pH 8 (1/20 volume)
Amount5 µL Concentration3 Molarity (M) ammonium acetate (1/10 volume)
Amount100 µL COLD 100% ethanol (2 volume)

• Mix well and incubate Temperature-20 °C for Duration02:00:00 to ensure plasmid precipitation
• Centrifuge Duration00:20:00 at maximum speed at Temperature4 °C
• Remove supernatant and add Amount500 µL 70% COLD ethanol
• Centrifuge Duration00:05:00 at maximum speed at Temperature4 °C
• Remove supernatant; resuspend in Amount10-20 µL RNase free H2O (based on the size of the pellet)
• Place the tube Duration00:05:00 at Temperature65 °C on heating block
• Measure DNA concentration
2h 30m
II. In vitro transcription with NEB Standart RNA Synthesis kit (E2040S):
II. In vitro transcription with NEB Standart RNA Synthesis kit (E2040S):
• Spin-down all reagents
• Keep the 10X Reaction Buffer at room temperature while assembling the reaction.
• Vortex all solutions until they are completely in solution.
• Assemble dNTP mix. (Amount per sample) Store the ribonucleotides on ice!

  • dATP (Concentration75 micromolar (µM) ) Amount2 µL
  • dCTP (Concentration75 micromolar (µM) ) Amount2 µL
  • dGTP (Concentration75 micromolar (µM) ) Amount2 µL
  • dUTP (Concentration75 micromolar (µM) ) Amount2 µL

• Assemble the reaction in an appropriate RNase free PCR tube:

1) Amount1 µg DNA plasmid (digested)
2) Amount8 µL NTP Mix
3) Amount2 µL 10X Reaction Buffer
4) Amount1 µL DTT
5) Amount2 µL T7 enzyme mix
6) RNase Free water up to Amount20 µL

  • Pipette the mixture up and down gently and microfuge
  • Incubate DurationOvernight at Temperature37 °C using a PCR machine. The shorter the ivtRNA, the longer the reaction time needed.
  • Template plasmid Digestion: add Amount70 µL nuclease free water, Amount10 µL of 10X DNase I buffer and Amount2 µL DNase I. Mix well, and incubate Duration00:15:00 at Temperature37 °C

6h 15m
III. RNA precipitation:
III. RNA precipitation:
Transfer to Amount1.5 mL RNase free tube and add:
  • Amount4 µL Linear acrylamide
  • Amount30 µL LiCl
Mix well, incubate Duration02:00:00 at Temperature-20 °C

• Centrifuge at 4°C/20 min/max
• Remove supernatant
• Add Amount1 mL 70% COLD ethanol
• Centrifuge at 4°C/5 min/max
• Remove supernatant and allow the pellet to dry
• Resuspend in Amount15-20 µL RNase-free H2O
• Place the tube Duration00:05:00 at Temperature65 °C water bath
• Dilute Amount1 µL in Amount9 µL H2O to measure concentration

2h 5m
Alternative to RNA purification: Monarch RNA Cleanup Kit. NEB #T2030
Before to start
  • The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.
  • Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.
  • Add 4 volumes of ethanol (>95%) to one volume of RNA Cleanup Wash Buffer

  1. Add Amount100 µL Cleanup Binding Buffer to the Amount50 µL sample. A starting sample volume of Amount50 µL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. For samples larger than 50 μL, scale buffer volumes accordingly. Samples with a starting volume > 150 μL will require reloading of the column during Step 3.
  2. Add Amount150 µL (1 volume) of ethanol (≥ 95%) to your sample and mix by pipetting or flicking the tube. Do not vortex. This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (300 μL) of ethanol to your sample instead of 1 volume (150 μL). The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.
  3. Insert column into collection tube, load sample onto column and close the cap. Spin for Duration00:01:00 , then discard flow-through. For diluted samples > 900 μL, load a portion of the sample, spin, and then repeat as necessary.
  4. Re-insert column into collection tube. Add Amount500 µL Cleanup Wash Buffer and spin for Duration00:01:00 . Discard the flow-through.
  5. Repeat wash (Step 4).
  6. Transfer column to an RNase-free 1.5 mL microfuge tube (not provided). Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for Duration00:01:00 to ensure traces of salt and ethanol are not carried over to the next step.
  7. Elute in Amount20 µL nuclease-free water.
  8. Place the tube Duration00:05:00 at Temperature65 °C water bath
  9. The eluted RNA can be used immediately or stored at Temperature-80 °C . Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency.

Lopez Lab Specifics: Characterization: To check QC (size and degradation) and endotoxins.
Lopez Lab Specifics: Characterization: To check QC (size and degradation) and endotoxins.
  • For QC

Person in charge: Michael Heinz mheinz@wustl.edu
4444 Forest Park Ave. 04140
Quantity: 30 ng in 10 μL nuclease free H2O.
Shipment: 1.7-2.0 mL microcentrifuge tubes / dry ice

  • For Endotoxins

Person in charge: Kelsi Rotter krotter@wustl.edu
Southwest Tower 00719
Quantity: 500 ng in 150 μL nuclease free H2O.
Shipment: 1.5 mL microcentrifuge (new plasticware certified to be pyrogen free) / dry ice
Protocol references
From plasmid DNA obtained by Promega PureYield Plasmid Miniprep System

Modified by Victoria Gnazzo from: IVT (in vitro transcription) STAR method_DF