Springer Lab README (readme.springerlab.org)

Here are some quick links and an FAQ for common Springer Lab tasks. (Edit this page)
Most of these links go to Springer Lab Documents on Google Drive or the Sysbio Intranet.
We also have a website at http://springerlab.org.

Strains & Inventory

Yeast (SLY) database

Plasmid (SLB/V) database. SLB are bacteria glycerol stocks in -80C. SLV are miniprepped plasmids in -20C. Plasmid maps are stored in a Geneious database (instructions for connecting).

Primer (SLD) database

Bacteria Strain Database (SLS) (Non-yeast, non-vector)

Strain collections (SLL) database. Including:

SLL1: Yeast deletion collection (MATalpha)
SLL2: Yeast GFP collection
SLL3: EUROSCARF plasmids (drug markers, fluors, etc)
SLL4: Liti/Fay natural isolates

SLL8, SLL08: TAP-Yeast Library Nov 2012

SLL9, SLL09: TetO library

SLL10: YEAST ORF Collection (Gal1 promotor) & gateway

SLL11: MoBY Clones for FLEXIQuant

SLL12: SGRP (Liti) Heterozygous Diploids
SLL13: Common lab strains (S288C) with different markers & fluors

SLL14: SGRP cerevisiae and paradoxus

SLL16: (the plate is gone)

SLL17: Liti/Fay natural isolates with GAL1pr-YFP

SLL18: EasyClone Yeast Toolkit (manual)

SLL19: MoClo Yeast Toolkit (YTK) (addgene, paper)

Enzyme Inventory. In a pinch, you can ask around at Paulsson, Silver, and Kirschner labs, who all have large enzyme collections.

Lab Logistics

Logins for online services and instrument computers

Logins of ordering systems for DARPA

Lab member contact info

Lab meeting schedule

Stratedigm / FACS core schedule

Journal club record

Reagent services

Ordering database. For ordering reagents and looking up previous orders. See here for orders before 5/14/2018.

Media prep. For ordering liquid and solid media. Choose from existing recipes or enter a new one.

Primers from IDT. For PCR, cloning, etc. See “logins” above for account info.

Sanger sequencing from Genewiz. Submission instructions here. See “logins” above for account info.

Sanger sequencing from Dana Farber. ~30% cheaper than Genewiz but less convenient. Use when you have many samples. PO #: 70001714374

Protocols

Colony PCR protocol

Yeast transformation protocol. Gietz lab via Jue.

Alternate yeast transformation protocol. Winston Lab via Christine. Uses TE to increase transformation efficiency. If you don’t know what this means, use the first protocol.

Setting up double gradients

Drug selection in yeast

Yeast nomenclature (SGD)

Random spore analysis

Springer Lab Molecular Cloning Protocols

Including Gibson Assembly, Golden Gate Assembly, Q5 Master Mix, OneTaq Master Mix, Ladder, Loading Dye, etc.

Springer Lab Yeast Protocols

Including yeast transformation protocols, transformation reagent recipes, yeast media recipes, TE buffer recipes, etc.

BASICS

I am new to the lab. What do I do?

• Ask to get access to the Springer Lab Google Docs and Google Calendar.
• Ask to be invited to the Springer Lab Slack team.
• Ask the Sysbio office to get an eCommons username so you can access Sysbio Intranet, Orchestra, software downloads, etc.
• Ask to be invited to the Springer Lab GitHub team.
• Ask to get put on the SPRINGERLAB Listserv email list.
• Ask lots of questions. If you get a good answer, add it to this FAQ to help others.

What software do I need? How do I get it?

• MATLAB for general data analysis. Free download from HMS research computing software or FAS downloads page.
• Geneious to manage DNA sequences. Also from HMS RC. See here for license information. See here for instructions to connect to the Springer lab Geneious database. You need to be on Harvard or HMS network to access the license server and database.
• TeamViewer to remotely access lab computers/equipment (Stratedigm). Has versions for Windows, Mac OS, iOS, and Android. Free download here.
• Microsoft Office. HMS buys licenses for us. See the Sysbio IT office on WAB 5th floor. Office is also part of the Office 365 For Harvard service. See here for instructions.
• Adobe Illustrator for making figures. Not free (ask Mike to buy for you).
• To manage papers, people use Mendeley (free), Papers (not free), or Paperpile (free via HMS site license).
• To keep a lab notebook, people use MS Onenote, Google Docs, MS Word, Benchling, or paper lab notebooks.

How to connect to HMS Private wifi?

• http://it.hms.harvard.edu/services/servers-network/wireless-networks

How to connect to Shared Departmental File Server?

• Address:

• Windows: \\research.files.med.harvard.edu\SysBio
• Mac: smb://research.files.med.harvard.edu/SysBio

• Username: MED\YourEcommonsID (Domain: MED. Username: YourEcommonsID)
• Password: YourEcommonsPassword
• Detailed instructions for Mac / PC.

WET LAB

Where can I get a strain with drug marker X, fluor Y, and mating type Z?

The master plate SLL13 (-80C freezer, rack “Springer Lab Plate Stocks”) contains strains with almost all combinations of {MATa, MATx} x {constitutive BFP, CFP, YFP, mCherry, or GAL1pr-YFP} x {kan, nat, hygro}, in either the BY4741/2 (isogenic to GFP or deletion collection) or FY4/5 (S288C prototrophic) genetic backgrounds. This plate also has WT stocks of BY4741, BY4742, BY4743, FY4, FY5, and F2.

How do I delete gene X?

If you don’t mind auxotrophies, you can get the deletion strain from our copy of the MATalpha (BY4742) yeast deletion collection. A MATa (BY4741) collection also exists in the Silver lab. To construct a deletion strain yourself, PCR-amplify a drug marker cassette with flanking homology to your gene of interest and do a yeast transformation. The most recent “standard” primer design uses 40bp of flanking homology, plus the S1/S2 priming sequences from Janke 2004. These will amplify drug marker cassettes from pFA6a-kanMX6 (D55V), pFA6a-natNT2 (D56V), or pFA6a-hphNT1 (D57V). The older “Pringle” or “Bahler” priming sequences are also widely used, but are only compatible with the kanMX cassette. Make sure to verify strains before using them (see below). This includes strains from the deletion collection, as they are not always correct.

How do I make a GeneX-GFP fusion?

If you don’t mind auxotrophies, you can get the GFP fusion strain from our copy of the yeast GFP collection (BY4741 background strain). These strains are described technically here and used in many papers. To construct a fusion yourself, amplify a fluorescent protein + marker cassette and transform it into your yeast. The most recent “standard” priming sequences are S3/S2 (C-term fusion) or S1/S4 (N-term fusion) from Janke 2004. These are compatible with pFA6 style plasmids containing a variety of fluorescent proteins, including recent ones from Lee 2013. The default FP to use is yEGFP (“yeast-codon-optimized Enhanced GFP”) (B11V in our plasmid collection); mRuby2 (D53V) is a good red option. Do not use the HO-TDH3pr-XFP vectors as a template for protein fusions, as they lack a linker sequence. Make sure to verify strains before using them (see below). This includes strains from the GFP collection, as they are not always correct.

How do I verify gene X deletion by colony PCR?

For a standard gene deletion (i.e. from the Yeast Deletion Collection or constructed using standard primers and marker cassettes as above), use the “ABCD colony PCR design” from the yeast deletion project. Follow this colony PCR protocol. For each deletion strain, do 2 “triplex” PCR reactions: one for the 5’ junction of the deletion cassette (primers GeneX_A + GeneX_B + kanB), another for the 3’ junction (primers GeneX_C + GeneX_D + kanC). As a control, include 5’ and 3’ triplex reactions on a WT strain. You can download ABCD primer sequences used in the yeast deletion project here. Our primer collection already has kanB/C and ABCD primers for commonly studied genes.

DATA ANALYSIS

What is O2?

O2 is the parallel computing cluster at Harvard Medical School. You should use it if you need to run many calculations in parallel, or if you are analyzing large datasets (like genome sequences). You can get an Orchestra account here, and view the documentation on O2 Wiki.

Where should I put my data on Orchestra?

The Springer Lab has reserved storage at the folder /groups/springer/. Make a subfolder for yourself and store your data/scripts there.

Where to get yeast genome sequences?

The Saccharomyces Genome Database hosts many tools based on the reference yeast genome (lab strain S288C). There are also many natural yeast strains, summarized here. We have recently analyzed many of these sequences, and the data & code related to this can be found on Orchestra at /groups/springer/sequences/.