Oct 04, 2024

Public workspaceX-region PolyU/UC mutagenesis

DOI
dx.doi.org/10.17504/protocols.io.8epv5rxy6g1b/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.8epv5rxy6g1b/v1
Protocol Citation: Carolina Lopez 2024. X-region PolyU/UC mutagenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rxy6g1b/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: October 04, 2024
Protocol Integer ID: 100753
Abstract
Protocol to PCR and in vitro transcribe the HCV X region w/out mutagenesis
Materials
STAR METHOD

Material:
Primer: X-region F/ X-region R (Concentration10 micromolar (µM) )
PolyU/UC F and R (Concentration10 micromolar (µM) )
dNTPs (Concentration10 millimolar (mM) ) Fermentas –For PCR
HCV PolyU/UC X-region plasmid
HCV X-region PolyU/UC PCR - T7 in vitro transcription
HCV X-region PolyU/UC PCR - T7 in vitro transcription
PCR:
1) Thaw component and cDNA, spin down before use
2) Assemble reaction: Master Mix: 10X (each reaction assemble 3 tubes)

Total: Amount50 µL

3) Run PCR


II. In vitro transcription:
II. In vitro transcription:
15m
15m
➢ 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 (2μL ATP+ 2μL CTP+ 2μL GTP+ 2μL TTP/reaction).
➢ Store the ribonucleotides on ice!

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) Amount0.5-1 µL RNaseOUT (RNase Inhibitor)
5) Amount2 µL T7 enzyme mix
6) RNase Free water up to Amount20 µL

• Pipette the mixture up and down gently and microfuge
• Incubate 3-6hrs at Temperature37 °C using PCR machine. The shorter the ivtRNA, the longer the reaction time needed.
• (Optional but do most times) Template plasmid Digestion: 1μLTurbo DNase and mix well, Duration00:15:00 at Temperature37 °C
15m
IV. RNA precipitation: Transfer to an 1.5ml RNase free vial
IV. RNA precipitation: Transfer to an 1.5ml RNase free vial
35m
35m
If you expect very low yields of RNA, do not dilute the transcription reaction with water prior to adding the LiCl.

• Add Amount30 µL H2O
Amount30 µL LiCl
• Mix well, incubate Duration00:30:00 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 Amount19 µL H2O to measure concentration
35m
Protocol references
Jie Xu (Lopez Lab)