Jul 11, 2024
Generating Ezrin Plasmids
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Generating Ezrin Plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32361v25/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: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103192
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Generating Ezrin Plasmids
1. pZac2.1-GfaABC1D-BioID2-HA Construction
- **Obtain Materials**
- pZac2.1-GfaABC1D-Lck-GCaMP6f plasmid from Dr. Baljit Khakh (Addgene plasmid #52924).
- pAAV-hSyn-BioID2-Linker-Synapsin1a-HA plasmid for BioID2 sequence.
- **PCR Amplification of BioID2**
- PCR amplify BioID2 sequence from pAAV-hSyn-BioID2-Linker-Synapsin1a-HA using the following primers:
- Forward Primer: 5’-ctagcctcgagaattcaccatgttcaaaaatcttatttg-3’
- Reverse Primer: 5’-ccgggtcgactctagatgcgtaatccggtacatcg-3’
- **Insertion into pZac2.1-GfaABC1D-Lck-GCaMP6f**
- Use In-Fusion cloning (TaKaRa) to insert the PCR-amplified BioID2 sequence into the EcoRI and XbaI restriction sites of pZac2.1-GfaABC1D-Lck-GCaMP6f.
2. pZac2.1-GfaABC1D-Ezrin WT-BioID2-HA and pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA Construction
- **Obtain Materials**
- pHJ421 (pEGFP-Ezrin WT) and pHJ423 (pEGFP-Ezrin T567D) plasmids from Stephen Shaw (Addgene plasmid #20680 and #20681).
- **PCR Amplification of Ezrin**
- PCR amplify Ezrin sequence from pHJ421 or pHJ423 using the following primers:
- Forward Primer: 5’-ctagcctcgagaattcaccatgccgaaaccaatca-3’
- Reverse Primer: 5’-tgaacatggtgaattccgacagggcctcgaactcg-3’
- **Insertion into pZac2.1-GfaABC1D-BioID2**
- Insert the PCR-amplified Ezrin sequence into the EcoRI restriction sites of pZac2.1-GfaABC1D-BioID2 for both Ezrin WT and Ezrin T567D variants.
3. pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA Construction
- **Mutagenesis**
- Use the Q5® Site-Directed Mutagenesis Kit (NEB) to generate the Ezrin T567A mutation in pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA.
- Perform mutagenesis using the following mutagenesis primers:
- Forward Primer: CAAGTACAAGGCGCTGCGGCAGA
- Reverse Primer: TCCCGGCCTTGCCTCATG
- **Confirmation**
- Verify the presence of the Ezrin T567A mutation in the plasmid through sequencing or restriction digest analysis.
Notes:
- Ensure proper sterile technique and use of appropriate safety precautions during handling of plasmids and reagents.
- Perform all steps under sterile conditions to avoid contamination.
- Validate all constructs through sequencing to confirm the correct insertion or mutation.