New Students

Welcome to the Norimatsu Lab and to the Cell Culture Project! This project is centered around culturing different types of cells, utilizing CRISPR technology, and for testing aspects of other projects with cultured cells. If you don't have a specific area of the project you want to start focusing on, it may seem overwhelming at first when searching through this section of the lab wiki. Below is a prioritized list (1 = first priority or a good starting point) of skills you can learn first to allow you to both assist with culturing cells as well as moving the CRISPR or other projects that utilize cells forward. These lists serve as a suggestion to help you get oriented to how the process of cell culture works and what the goals of using our cells for CRISPR/other projects are.

Cell Culture

1. Read about and understand sterile technique. Sterile technique is required to be implemented for every cell culture protocol, and failing to do so can easily lead to cell death. Information about sterile technique in our lab can be found here, and the protocol can be found on the cell culture protocols page.

2. Read about and learn how to passage cells. Background information for passaging can be found here, and the protocol for passaging cells can be found on the protocols page. Passaging cells is imperative to keeping the cell culture alive, and having as many people as possible available to passage can ensure someone is always able to passage the cells when needed.

3. Read about and learn how to pre-plate cells for transfection, as well as how to transfect DNA into mammalian cells. Transfections allow us to introduce foreign DNA into the cell so that we can measure the effects of doing so. Information about transfections can be found here, and protocols for how to pre-plate cells for transfection and how to transfect cells can be found on the protocols page.

4. Read about and learn how to use the AmScope microscope camera to observe cells, as well as how to observe cells under fluorescence. Many of the plasmids we will be transfecting will contain some type of fluorescence protein (usually GFP) that will cause the cells to fluoresce a certain color in response to a specific type of light. We can use this fluorescence as a way to report or measure results, and the AmScope serves as a way to achieve higher quality images of the cells. Information about observing cells under fluorescence can be found here, and protocols for how to observe the cells under fluorescence and how to use the AmScope can be found on the protocols page.

5. Read about and learn how to freeze cells for long term storage. We currently use CryoStor freezing media to freeze stocks of each cell line we are working with in the -80C freezer for use in the future. Currently we only freeze cells as needed to maintain a stock of a few frozen tubes of each cell type. Information about why and how we freeze cells can be found here, and protocol for freezing cells can be found on the protocols page.

6. Read about and learn how to thaw cells freezing at -80C. We generally only thaw cells when attempting to restart a cell line and as long as the cell cultures remain healthy it will not need to be done often. Therefore, learning this is likely your lowest priority unless cells need to be actively thawed. More information about thawing cells can be found here, and a protocol on how to do so can be found on the protocols page.

This pdf from Invitrogen outlines the basic concepts and protocols of nearly all cell culture techniques and concepts used in this lab. You do not need to memorize all of the information, but it can be an additional resource to reference when wanting to know more about a certain protocol or need help understanding a term.

CRISPR

1. Read about and establish a basic understanding of CRISPR Cas9. We use a variation of this system called "prime editing 3.0", or "prime editor 3", but understanding how the standard CRISPR Cas9 system works first could help with understanding prime editing. Google slide and zoom presentations of CRISPR Cas9 can be found on the google drive, and the CRISPR page outlines a basic understanding of CRISPR. An article outlining the history and development of CRISPR techologies can be found here.

2. Read about and work to understand the different types of CRISPR variations, such as Prime Editing and Base Editing. More information about Prime Editing and Base Editing can be found on the CRISPR page.

3. Read about desiginging base editing and pegRNA systems. This would likely involve learning how to design base editing guide RNA for the desired edit and/or learning how to design prime editing guide RNA (pegRNA). pegRNA is an essential component of PE3, and specific PE3 will have to be designed for each edit we wish to perform. One tool we can use to design pegRNA is called "PegFinder". More information about base editing and pegRNA can be found under the Prime Editing section of the CRISPR page, and protocol for using pegFinder to design pegRNA can be found on the cell culture protocols page.

4. Once we have an edit picked out and pegRNA designed (for prime editing), or a system designed for base editing, we have to design and purchase a plasmid to transfect into the target cells. This plasmid will need to contain sequences that will encode and allow for transcription of our base editing components (base editor Cas9 + gRNA) or prime editing components (prime editing protein complex + pegRNA). Some additional info can be found on the designing the plasmids page, but additional info wil likely need to be added.