Jul 11, 2024
Generating pPB-CAG-mCherry-CAAX Plasmid
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Generating pPB-CAG-mCherry-CAAX Plasmid. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q34pl1y/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: 103191
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Generating pPB-CAG-mCherry-CAAX Plasmid
**Obtain Plasmids** - Obtain pPB-CAG-EGFP and pGLAST-PBase plasmids from Dr. Joseph Loturco.
**Insert mCherry-CAAX into pPB-CAG-EGFP** - Insert mCherry-CAAX between XmaI and NotI restriction sites in pPB-CAG-EGFP to replace EGFP.
**Insert hU6 Promoter and shRNA into pPB-CAG-mCherry-CAAX** - Amplify a DNA fragment containing hU6 promoter and shRNA from pLKO.1-shRNA using Phusion High-Fidelity DNA Polymerase with primers introducing SpeI restriction sites: - Forward Primer: GGACTAGTCAGGCCCGAAGGAATAGAAG - Reverse Primer: GGACTAGTGCCAAAGTGGATCTCTGCTG - Purify the PCR products and digest them with SpeI.
**Ligation into pPB-CAG-mCherry-CAAX** - Ligase the purified and SpeI-digested DNA fragment containing hU6 promoter and shRNA into pPB-CAG-mCherry-CAAX at the SpeI restriction site.
**Confirmation by Analytical Digest and Sequencing** - Perform an analytical digest with EcoRI to confirm the correct orientation of the inserted DNA fragment. - Sequence the plasmid to confirm the accurate insertion of hU6 promoter and shRNA sequences.