Keeping a detailed plasmid inventory (with info about the plasmid itself (sizes, antibiotic resistance, promoters, etc.) as well as the protein you want to make from it (sizes, pI, UV absorption, with & without tag, etc.) can save you a lot of time digging through notes and re-researching things to find the info you need. So I hope this quick demo of what I’ve found helpful will hope you succeed!
You can download my template here: https://docs.google.com/spreadsheets/d/1eMbZUuWlqO-CEYcR445ZaIHUnZYg2uwq/edit?usp=sharing&ouid=110501489579733284162&rtpof=true&sd=true
And here are some hopefully helpful links:
a good review article: Waugh D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags. Protein expression and purification, 80(2), 283–293. https://doi.org/10.1016/j.pep.2011.08.005
More on various proteases: https://www.emdmillipore.com/US/en/life-science-research/protein-sample-preparation/protein-purification/cleavage-enzymes/0Uqb.qB.V5gAAAFBOFJlvyyv,nav
more on TEV sequence requirements: Kapust, R. B., Tözsér, J., Copeland, T. D., & Waugh, D. S. (2002). The P1′ specificity of tobacco etch virus protease. Biochemical and biophysical research communications, 294(5), 949–955. https://doi.org/10.1016/S0006-291X(02)00574-0
Early Career Researcher Toolbox: Free Online Molecular Biology Tools, By Beth Kenkel 2020 https://blog.addgene.org/free-online-molecular-biology-tools
ExPasy Translate: https://web.expasy.org/translate/ ExPasy
ProtParam: https://web.expasy.org/protparam/ ApE, A plasmid Editor by M. Wayne Davis: https://jorgensen.biology.utah.edu/wayned/ape/
plasmid repository links:
- Addgene: https://www.addgene.org/
- DNASU: https://dnasu.org/DNASU/Home.do
random other helpful tools for a variety of things: https://bit.ly/bumblingbiochemisttoolbox
More on cloning methods here: http://bit.ly/molecularcloningguide
more about sub cloning and various plasmids: blog form: https://bit.ly/vector_plasmids ; YouTube: https://youtu.be/2vLTz0lPlFI
