Guidelines for materials, equipment, reagents, strains, in the wet lab space.

This document describes where we keep things in the wet lab.

It also describes how we keep track of new materials that we generate, such as DNA, plasmids, and yeast strains.

It’s important that before you begin any work in the wet lab, you have access to the Datastore – please see newstart page in the lab manual for information on how to set this up.

Inventory - everything we have in the lab

The inventory is stored on the Datastore using the following filepath csce.datastore.ed.ac.uk/csce/biology/groups/wallace_rna/admin/Inventories/Consumables/Wallace_Lab_Inventory.xlsx.

This spreadsheet contains details on everything that we have in the lab, split into 5 separate tabs; Consumables, Reagents, Antibodies, Chemicals and Equipment. There’s a column for item name, source (meaning the company that it is ordered from), catalogue number (important for reordering the item on Sciquest), quantity (how many were purchased at each time) and storage location. There’s also a separate column for comments on any of the items i.e, out of stock, long lag time etc.

Inventory ordering and tracking

This inventory sheet doubles as our order tracking system. Currently, the bulk of the ordering is carried out by Liz Hughes. If you order your own reagents/consumables, please specify what you are ordering in the “Orders” channel on Slack, and add details on to the spreadsheet. If you do not, please give details of what you would like to order, catalogue number and quantity on the “Orders” channel on Slack.

After an item has been ordered, it will be highlighted in red on the inventory spreadsheet. If you are in the lab when the order comes in, please make sure to put the item in its correct location, and once you have done this please turn the text for this item back to black on the inventory spreadsheet. If you are unsure about storage of new items, please check the MSDS sheet if applicable, or ask another lab member to help you out with this.

Strains, Plasmids, etc.

For DNA and plasmids, everyone has space in our -20ºC freezer in room 2.19 to store working stocks for daily use. We also have a lab fridge in room 2.19, where bacterial/yeast plates can be stored in the short-term whilst experiments are ongoing.

The lab’s main database of strains and plasmids is on datastore at wallace_rna/inventories/WallaceLab_StrainsPlasmids.xlsx.

The importance of long-term stocks

It is essential to have long-term stocks of all plasmids (stored in bacteria/yeast), and all edited S. cerevisiae and C. neoformans strains. It is essential to have a backup copy in case catastrophe strikes - a failed freezer or a building fire. It is also essential to track the data about strains in a consistent format. This is all essential, because future you, others in the lab, and collaborators, need to be able to rely on the strains and build on your results. For strains that make it to a publication, those strains need to be available to send to other researchers for years into the future.

Please ask on the #lab channel on slack if there is any doubt at all about any of this.

Guidelines for making and storing glycerol stocks

Glycerol stocks at -80ºC store yeast and bacteria indefinitely.

When making new strains, it is worth making temporary glycerol stocks of a number of clones from your transformations while you verify them so that they do not acquire additional random mutations. These can be made in sterile Eppendorf tubes by mixing 100ul of o/n culture in the relevant growth medium (with selection if necessary) with 100ul 30% glycerol in water or growth medium and stored in your own space in the -80ºC Freezer. Once verified you should streak these stocks out on relevant growth media plates, grow up o/n cultures and make duplicate stocks of each strain in cryo-tubes by mixing 900ul of o/n culture with 900ul 30% glycerol in water or growth medium, put a cap in the top of each tube and label both the cap and the side of the tube with an indelible marker pen. One tube should go into each of the 2 relevant strain boxes (yeast/ Crypto/ E.coli (Set A and B)) in Shelf1/StackA(SetA) and Shelf2/StackA(SetB). For many mutant strains, e.g. CRISPR mutants, it is worth freezing more than one independent transformant of the strain if you have them – keep the same strain name but add a suffix (eg. yRB002.2 and yRB002.3).

A printable version of this protocol is on protocols.io.

TODO: make plans for backup box of strain and plasmid collection to be stored in different building.

Yeast strain information to record in lab database

For yeasts (Saccharomyces, Cryptococcus, Candida, etc.), you need to systematically record full details of the strain.

• Name - id of strain, e.g. the second strain of yeast made by Rosey Bayne is yRB002.
• Alias - short descriptive name for strain, (eg. PAB1-Clover).
• Genotype - systematic description of yeast genotype, as described in table of yeast genetic nomenclature, taken from Getting Started With Yeast, Sherman 2002.
• Relevant Genotype - the key genes or mutations that the strain is used for.
• Comments - useful information about the strain and any other relevant info (you can get an idea of what is required by looking at existing records).
• Parent Strain - the id of strain that was the parent of the transformed strain. This should be identical to the id in the lab database. For external strains, this may be found in the publication reference; for some strains it may not be relevant.
• Source - your name if you made it or who/where you got it from.
• Growth Medium - including antibiotics where relevant.
• Date - date entered to database
• Box - Box number where strain stored
• Position in Box - location of that tube
• Reference - to publication, if relevant; including doi.
• Links - links to relevant files in datastore. This would usually be a snapgene .dna file describing the set of transformations made to the locus of interest.

Examples

TODO: give a couple of full-record examples.

Plasmid information to record in lab database

Most plasmids are stored long-term in E. coli bacterial strains.

• Name - id of plasmid
• Alias - plasmid description
• Comments - information about the strain - (eg. “this strain is dam+ so plasmid isolated from it will not cut with BclI. You need to transform the plasmid into a dam- strain before isolating plasmid to clone gRNAs but dam- strains are unstable so not suited to making frozen stocks””)
• Host strain - the bacterial strain hosting the plasmid - (eg. ccdb, TOP10, NEB5alpha, etc.)
• Source - your name if you made it or who/where you got it from.
• Media - growth medium and relevant antibiotic fto maintain selection.
• Date - date entered to database.
• Box- Box number where strain stored.
• Position - location of that tube in the box.
• Link to plasmid map - link to plasmid map in datastore, in either snapgene .dna format or genbank .gb format. Ideally the filename of this plasmid map should begin with the plasmid name, such as pEW001_my_first_plasmid.dna

Please refer to “Where data belongs” section of lab manual for more information on file organization on datastore.