Generating pPB-CAG-mCherry-CAAX Plasmid
Shiyi Wang
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Generating pPB-CAG-mCherry-CAAX Plasmid
Steps
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.
