Last updated August 2023.
Here are a list of online resources that should help you find information about DNA part sequences, molecular biology questions or basic protocols for handling DNA/RNA/proteins and E. coli, and to help you to begin to troubleshoot your own experiments. Most resources are freely available. Note that this list isn't meant to be exhaustive, it is mainly to get you started.
- Bionumbers - https://bionumbers.hms.harvard.edu/search.aspx
- Calculators - conversions http://www.genscript.com/conversion.html, https://labhacks.net/#calculators
- PCR calculator - https://ec363.shinyapps.io/pcr_calc/
- Lab Hacks app - https://apps.apple.com/us/app/lab-hacks/id1462593060#?platform=iphone
Codes and abbreviations
- Nucleotide abbreviations - https://en.wikipedia.org/wiki/Nucleotide#Abbreviation_codes_for_degenerate_bases
- DNA modifications (IDT) https://eu.idtdna.com/site/Catalog/Modifications/
- Modified dNTPs (and similar) - https://www.jenabioscience.com/nucleotides-nucleosides/nucleotides-by-structure
- Amino acids (abbreviations and properties) - https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#Chemical_properties
- Codon Table - https://en.wikipedia.org/wiki/DNA_codon_table
Codon usage tables
- E. coli and others
- K-12 http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=83333
- B http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=413997
- W3310 http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=316407
- all E. coli http://www.kazusa.or.jp/codon/cgi-bin/spsearch.cgi?species=escherichia+coli&c=i
- K-12 http://openwetware.org/wiki/Escherichia_coli/Codon_usage
- unspecified strain of E. coli: https://www.genscript.com/tools/codon-frequency-table
- Rare codon analysis tool- https://www.genscript.com/tools/rare-codon-analysis
Tools
- Clustal Omega, DNA/RNA/Protein alignment http://www.ebi.ac.uk/Tools/msa/clustalo/
- Blast, sequence search in databases http://blast.ncbi.nlm.nih.gov/Blast.cgi
- Back-translation http://www.ebi.ac.uk/Tools/st/
Buffers
- Buffers for biochemical reactions -
- https://www.promega.co.uk/resources/product-guides-and-selectors/protocols-and-applications-guide/buffers-for-biochemical-reactions/
- http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
- http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html
Protocol databases
- Addgene protocols (molbio and cloning) - https://www.addgene.org/protocols/
- Addgene guides - https://www.addgene.org/guides/
- Addgene ebooks - https://www.addgene.org/educational-resources/ebooks/
- Addgene Plasmids 101 blog - https://blog.addgene.org/topic/plasmids-101
- Protocols.io - https://www.protocols.io/
- CSHL Protocols (mainly molbio) - http://cshprotocols.cshlp.org/site/misc/subject.xhtml
- OpenWetWare - http://openwetware.org/wiki/Protocols
- Barrick Lab protocols (microbiology) - https://barricklab.org/twiki/bin/view/Lab/ProtocolList
Protocol FAQs
- ResearchGate Forum (Sign up to let you browse Q&As better) - https://www.researchgate.net/topics
- Introduction to recombinant DNA technology by Addgene https://www.addgene.org/mol-bio-reference/
- Enzymes used in molecular biology: a useful guide https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570007/
- Why Johnny can't clone: Common pitfalls and not so common solutions https://www.future-science.com/doi/10.2144/000114324
Software:
- Benchling (free) - Labbook and DNA analysis package. https://www.benchling.com/
- Snapgene (£££) - DNA analysis package. http://www.snapgene.com/support/tutorial_videos/introduction_to_snapgene/
Plasmids:
Plasmid databases:
- commercial
- donated https://www.addgene.org/browse/
- other https://dnasu.org/DNASU/Home.do , https://dnasu.org/DNASU/SearchOptions.do?tab=1
Plasmid parts: The vector
- What is a plasmid https://blog.addgene.org/plasmids-101-what-is-a-plasmid
- Origin of replication http://blog.addgene.org/plasmid-101-origin-of-replication
- Antibiotic resistance gene - https://blog.addgene.org/plasmids-101-everything-you-need-to-know-about-antibiotic-resistance-genes
- Antibiotic working stocks - https://www.addgene.org/mol-bio-reference/antibiotics/
- though Cam is often used at 34µg/ml final, slightly more than 25 µg/ml.
Plasmid parts: The construct
- Databases and lists of common parts:
- iGEM Registry databases and distributions: http://parts.igem.org/assembly/libraries.cgi.
- Sequences of common parts
- Snapgene -> Detect common features
- Addgene's list https://www.addgene.org/tools/reference/plasmid-features/
- Promoters https://blog.addgene.org/plasmids-101-the-promoter-region
- Andersen promoters (set of constititive promoters from weak to strong): iGEM pages.
- RBS design calculators
- Common Epitope Tags
Cloning methods:
- Types of cloning
- Traditional cloning (Type II restriction digest)
- Type IIS cloning combined with PCR cloning (= recommended as default)
- Oligo cloning
- Gibson assembly (also recommended)
- SPLiCE (by Edo Gianni) - http://openwetware.org/wiki/SPLiCE
Primer design:
- General rules on primer design (but keep Tm differences to 3oC max)- https://www.addgene.org/protocols/primer-design/
- Primer analysis
- Snapgene has Tm calculations that work OK.
- IDT is more thorough: https://eu.idtdna.com/calc/analyzer
- with formulae: http://www.genscript.com/cgi-bin/tools/primer_calculation
- Primer design for annealed oligo cloning https://www.addgene.org/protocols/annealed-oligo-cloning/
DNA sequence optimisation tools:
- Synonymise- small R tool I wrote to randomise codons where repetitive codon usage was not desired- https://github.com/ec363/synonymise
- DNA Chisel- Python tool for avoiding homologies, tuning GC content, codon optimisation- https://github.com/Edinburgh-Genome-Foundry/DnaChisel
E. coli strains:
- E. coli strains for cloning: https://www.addgene.org/mol-bio-reference/#strains
- NEB 10beta- https://www.neb.com/products/c3020-neb-10-beta-electrocompetent-e-coli
- NEB T7 express lysY/lacIq- https://www.neb.com/products/c3013-t7-express-lysyiq-competent-e-coli-high-efficiency
- BL21(DE3)- https://www.neb.com/products/c2527-bl21de3-competent-e-coli
- More strains: https://www.addgene.org/mol-bio-reference/strain-information/
- Common lab strains: https://blog.addgene.org/plasmids-101-common-lab-e-coli-strains
General cloning protocols:
Essential basic protocols:
- Overview https://www.addgene.org/protocols/
- General Cloning Protocols:
- Use these for basic principles only. Always follow reagent-specific instructions for reaction mixture recipes and thermocycler protocols
- PCR https://www.addgene.org/protocols/pcr/
- Restriction digests https://www.addgene.org/protocols/restriction-digest/
- DNA analysis by agarose gel electrophoresis https://www.addgene.org/protocols/gel-electrophoresis/
- DNA ligation https://www.addgene.org/protocols/dna-ligation/
- Bacterial transformation using electroporation: see wet-lab-protocols
- Bacterial transformation using heat shock https://www.addgene.org/protocols/bacterial-transformation/
- Overnight bacterial cultures https://www.addgene.org/protocols/inoculate-bacterial-culture/
- Creating glycerol stocks https://www.addgene.org/protocols/create-glycerol-stock/
- Streaking a plate from a glycerol stock https://www.addgene.org/protocols/streak-plate/
- Minipreps: DNA purification from bacterial culture https://www.addgene.org/protocols/purify-plasmid-dna/
- Quantification of DNA using a Nanodrop/spectrophotometer https://www.addgene.org/protocols/dna-quantification/
- Sequence analysis of DNA https://www.addgene.org/protocols/sequence-analysis/
- More:
- Phenol-chloroform extraction and ethanol precipitation https://www.addgene.org/protocols/purify-plasmid-dna/
Reagent and kit-specific information:
- Reagent-specific information can be found on the manufacturer's website.
- Kits: Minipreps, PCR purification, Gel extraction - All include manuals within the boxes, which can also be found online.
More information:
- Typical DNA marker ladders:
- Restriction enzymes:
- SnapGene, EnzymeX, NEB website all have good info on these
- https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes
- Specific PCR protocols
- Sequencing
- Follow instructions on provider's webpage about how to prepare samples. They often have stock primers for standard plasmids.
- Protocols for automating DNA assembly with OpenTrons: https://github.com/BASIC-DNA-ASSEMBLY/DNA-BOT
- Commandments of experimental microbiology (Barrick) https://barricklab.org/twiki/bin/view/Lab/StandardMicrobiologicalPractices
- OD600 calculator http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp
E. coli strains/genotypes:
- List of strain genotypes https://openwetware.org/wiki/E._coli_genotypes
- What the genotypes mean http://openwetware.org/wiki/Escherichia_coli/Nomenclature_%26_Abbreviations
- List of E. coli strains, but no links: http://www.uniprot.org/taxonomy/83333
E. coli genome/genes:
- EcoGene (when it was live it was great) http://www.ecogene.org/
E. coli general:
- openwetware (protocols): http://openwetware.org/wiki/Escherichia_coli
- wiki: http://ecoliwiki.net/colipedia/index.php/Welcome_to_EcoliWiki
- strains http://ecoliwiki.net/colipedia/index.php/Category:Strains
- gene lists http://ecoliwiki.net/colipedia/index.php/Category:Gene_Lists
- genomes: http://ecoliwiki.net/colipedia/index.php/Category:E._coli_genomes
- DH10b
- BL21 (DE3)
- EcoCyc: http://ecocyc.org/
- literature-based curation of the K12-MG1655 genome
- Characteristics of the E. coli genome (Barrick) https://barricklab.org/twiki/bin/view/Lab/ReferenceEColiGenome
Optimisation (maximisation):
- Genscript's description is an overview of some considerations for maximisation & includes both mammalian and bacterial elements https://www.genscript.com/codon-optimization-for-increased-protein-expression.html
Protein electrophoresis:
- typical protein ladder: https://www.neb.com/products/p7712-color-prestained-protein-standard-broad-range-11-245-kda
- hall of shame: http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html
- Tricine-SDS-PAGE for small proteins and peptides <30 kDa (especially if <15 kDa, as 15-30 can just about be resolved by standard glycine/Laemmli-SDS-PAGE). This paper is great resource not just for Tricine gels but also for hydrophobic proteins, membrane proteins, SDS, DTT/beta-merc reducing agents, etc. 2006 Schägger et al. Nat Protocols doi:10.1038/nprot.2006.4
Troubleshooting:
- EMBL https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/index.html
- RBSCalculators https://salislab.net/software/
Cell lysis and clarification:
- EMBL https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/index.html
- EMBL - E. coli lysis w sonication https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/sonication/index.html
- EMBL - E. coli lysis w lysozyme https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/enzymatic_lysis/index.html
- Wolfson centre protocols http://wolfson.huji.ac.il/purification/Purification_Protocols.html
Purification techniques:
- Common Epitope Tags - https://www.addgene.org/mol-bio-reference/#tags
- Cytiva Handbooks on Principles & Methods in Protein Purification (previously GE Lifesciences/Amersham Biosciences) https://www.cytivalifesciences.com/en/gb/support/handbooks
- EMBL https://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index.html
Concentration analysis:
- Comparison (Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem). http://wolfson.huji.ac.il/purification/Protocols/Comparision_of_Methods.htm
- A280 ext coeff: https://www.expasy.org/resources/protparam
Storage:
Protein structure databases
- UniProt http://www.uniprot.org/
- PDB (Europe) http://www.ebi.ac.uk/pdbe/
- RCSB http://www.rcsb.org/pdb/home/home.do (Research Collaboratory for Structural Bioinformatics, US)
- Others (list) - https://en.wikipedia.org/wiki/Protein_structure_database of interest:
- Proteopedia: essentially a wikipedia for proteins.
- ProtCID: Protein common interface database (for homologous proteins).
Amino acid properties
- http://www.sigmaaldrich.com/life-science/metabolomics/learning-center/amino-acid-reference-chart.html
- https://en.wikipedia.org/wiki/Amino_acid
- https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#Chemical_properties
Protein Properties
- Protein properties, Expasy http://www.expasy.org/resources
Protein families by structure
- Protein-Ligand binding: online simulation of the fraction of protein molecules in ligand-bound state as a function of binding affinity, protein concentration, and ligand concentration https://binding.streamlit.app
- SMILES (chemistry notation) generator https://www.cheminfo.org/flavor/malaria/Utilities/SMILES_generator___checker/index.html
- Small molecule permeability in Gram negative bacteria http://www.entry-way.org/pages/about
- FPbase: database of fluorescent proteins https://www.fpbase.org
- Introduction to experimental design (Barrick) https://barricklab.org/twiki/bin/view/Lab/IntroductionToExperimentalDesign
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R. Recommended over Excel for data analysis and presentation. A guide to downloading R/RStudio and getting started using R is provided on the Coding Resources page: https://github.com/ec363/coding_resources
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GitHub. Again, see the Coding Resources page: https://github.com/ec363/coding_resources
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Paper on Tidy Data (Wickham, 2014) https://www.jstatsoft.org/article/view/v059i10
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Parsley: Our web application for parsing data from exported format data from plate readers. Parsing = data extraction + data tidying + metadata joining. App and guide are here: https://gbstan.shinyapps.io/parsleyapp/ and preprint is here https://zenodo.org/record/8072500.
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FPCountR: Our R package for the conversion of fluorescence measurements from microplate reader data from 'arbitrary units' to absolute units (ie. molecules of fluorescent protein), and the analysis of timecourse E. coli data from 'fluorescence/OD' to 'molecules/cell or molar concentration'.
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Growth curve analysis with R. https://github.com/sprouffske/growthcurver
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Image analysis with ImageJ https://imagej.net
- How to prepare figures (not a bad set of guidelines) https://barricklab.org/twiki/bin/view/Lab/ProtocolsGraphGuide
Photos and Diagrams:
- Unsplash (free) - source of free stock photos that work well for presentations. https://unsplash.com/
- Biorender (£££) - scientific illustration application in the browser. expensive but excellent. https://app.biorender.com/
Data visualisation: