path: root/diybio/faq/better-questions.mdwn

This is a scratchpad for selecting better questions to include in a biohacking FAQ. The previous version of the FAQ seems to be inadequate for answering the most common questions from the [DIYbio mailing list](http://groups.google.com/group/diybio).

[[!toc  levels=4]]

# Segmentation and clustering

Biology is a really very tremendously huge topic, and choosing a good way to cluster questions might mean less effort in maintaining these FAQs in the future. Here is one possible way to organize the questions.

* Splitting it up into a bunch of smaller topics:
    * biology fundamentals
    * gel electrophoresis questions
    * transformation questions
    * incubation
    * all things glowing/gfp
    * thermocycling/pcr
    * dna extraction
    * sequencing
    * primers and primer design
    * stochiometry/dilutions/buffers/math
    * making equipment
    * buying equipment
    * software questions
        * biopython/bioperl/biojava/bioruby/biowhatever
        * plasmid design
        * primer design
        * file formats
        * sequence alignment
        * compression algorithms
        * biobricks
        * sbml and libsbml
    * basic questions about diybio as a community
    * diybio ethical and moral questions
    * safety and biosafety questions
    * security questions
    * legal questions

# Question dump

* I am interested in your projects and I would love to participate and help in any way possible.

* Where do I start?
    * If you are completely new to biology, start with a <a href="#books">book</a>.

* How do I get my project started?

* What knowledge is required to perform genetic manipulation experiments?

* What are some resources for DIY biology?

* What are some resources for not-necessarily-biology DIY projects?

* What sort of biohacking projects are possible?

* What does a basic lab setup require?

* As a high school student, what biology projects can I do?

* Has anyone made a new lifeform in their garage?

* What does a garage biology lab look like?

* What is the goal of do-it-yourself biology?

* Are there historical precedents or prior cases which have demonstrated similar issues to DIYbio?

* What is a biohacker?

* What is hacking?

* Where can I see an archive of previous diybio discussions?

* What are the capitalization rules for this stuff? Is it DIYbio, DIYBio, DIYBIO, diybio, diyBio, or DIY-BIO?

* Who owns diybio.org?

* Is there a biohacking group in my area?

* What's the difference between DIYbio, biohacking and biopunk, if any?

* What is open source software?

* What is open source hardware?

* Is DNA/RNA really like Software?

* I am a programmer: what is the "hello world" of synthetic biology or genetic engineering?

* What is "open access"?

* What is "open source"?

* What is "Creative Commons"?

* Which community college classes are relevant?

* Should I join a molecular biology lab on a college campus?

* What is a hackerspace?

* What is BioCurious?

* What is Genspace?

* Is there a local group in my area?

* How do I start a biohacking group in my city?

* What happens at a hackerspace, at Genspace, at BioCurious or at a local biohacking group?

* How can I join an academic molecular biology lab?

* What is Escherichia coli? Is it safe? Why is it used?

* How can I make Escherichia coli glow?

* What is a plasmid?

* Where do I get or order plasmids?

* Where do I get or order cultures?

* What is microbiology?

* What is molecular biology?

* What is synthetic biology?

* What is genetic engineering?

* What are the differences between synthetic biology and genetic engineering?

* What is bioinformatics?
    * <http://bioinformatics.org/wiki/Bioinformatics_FAQ>
    * The original version of the bioinformatics.org FAQ was written by "Damian": <http://bioinformatics.org/w/index.php?title=Bioinformatics_FAQ&oldid=1367>

* What are some recommended books for bioinformatics?
    * books:
        * <http://bioinformatics.org/wiki/Recommended_books>
        * <http://bioinformatics.org/wiki/Books>
        * <http://compbiology.org/?section=books>
        * <http://compbio.berkeley.edu/people/brenner/misc/books-compbio.html>
        * <http://www.brc.dcs.gla.ac.uk/~actan/bioinformatics/BioinformaticsBooks.html>
        * [Molecular Biology of the Cell](http://www.ncbi.nlm.nih.gov/books/NBK21054/) <http://www.ncbi.nlm.nih.gov/books/NBK21054/>

* What open source software for bioinformatics exists?

* Where can I download genomes?
    * <http://ftp.ncbi.nlm.nih.gov/genomes/>

* What is a homologue?
    * ["Homology" is a much-misused term](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3621342&dopt=Abstract) and existed in biology long before the notion of protein sequences. Strictly homology cannot be qualified; it is not correct to state that two proteins are "30% homologous" with each other, for example. If we could look back far enough in the evolutionary histories of any two molecules under comparison, we would be guaranteed to find a common ancestor eventually, but this is not true homology. An example of this would be the relationship between two variants of a single ancestral enzyme resulting from a gene duplication event. As a rule-of-thumb, true homology should be assigned only when the feature which leads us to suspect a relationship between molecules is one we consider likely to have derived from the molecules' common ancestor. To quote Page and Holmes [Molecular Evolution: A Phylogenetic Approac, Roderick D. M. Page and Edward C. Holmes; Blackwell Scientific; ISBN 0865428891]: "The classic molecular example is the parallel evolution of amino acid sequences in the lysozyme enzyme in leaf-eating langur monkeys and in cows. Both animals have independently evolved foregut fermentation using bacteria, and in both cases lysozyme has been recruited to degrade these bacteria. Therefore, langur and cow lysozymes are homologous as genes; however, as digestive enzymes they are not homologous because this functionality was not present in the ancestral lysozyme." Although sequence determines structure, it is possible for two proteins to have very different sequences and functions and share a common fold. In fact, most gene products with similar three-dimensional structures are insufficiently similar at the sequence level for true homology or analogy (non-homologous similarity) to be distinguished

* How can I tell if my cool idea is realistic, practical, possible, or completely impossible?

* How do I edit this wiki?

* How can I get access to scientific papers, journals, and pdfs?
    * join your local community college, pay for a single class and get their library card and access journals through [[ezproxy]]
    * send an email/request to [diybio@googlegroups.com](mailto:diybio@googlegroups.com)
    * <http://reddit.com/r/scholar>

* What is ezproxy?

* Where do you purchase lab equipment?

* How do I tell if lab equipment is in working condition?

* Why does commercial lab equipment cost so much?

* Where do you purchase reusable reagents?

* Which suppliers are DIY-friendly?

* What projects or experiments cost less than $100.00?

* What projects or experiments cost less than $1,000.00?

* What projects or experiments cost less than $10,000.00?

* What can I do with genetic engineering?

* What is a lab protocol?

* Where can I find lab protocols?

* What are some advanced lab techniques or procedures?

* Which lab techniques are important?

* Which microscope should I buy?

* What is a sub-stage condenser?

* Do I want a sub-stage condenser in a microscope?

* Does high magnification get me more information in a microscope?

* Is it better to get a high numerical apperature microscope or a high magnification microscope?

* What is phase contrast?

* What is a microarray?

* What is luciferin?

* Which bacteria are easy to work with?

* How do I get DNA into bacteria?

* How does electroporation work?

* What is gel electrophoresis for?

* What is a DNA or gel ladder?

* How deep are electrophoresis gels?

* How can I make a cheap gel for electrophoresis?

* How do I analyze a gel?

* What is the relationship of the FBI with DIYbio?

* What should I say to this journalist that keeps calling me?

* Who could I tell a journalist to speak with instead of me?

* When I transfer bacteria and put them into an exponential growth phase, what temperature should I keep?

* Can I use a water bath to incubate bacteria during a transformation protocol?

* What are the legal issues regarding obtaining ampicillin or other DEA scheduled antibiotics for research use?

* What are the legal issues regarding obtaining hormones or amines like norepinephrine or dopamine for research use?

* How well does autoclaving work?

* Can I use a microwave oven to sterilize a culture?

* Do microwaves damage DNA?

* Does ultraviolet radiation damage DNA?

* How does UV damage DNA?

* What concentration do I need for a CaCl2 transformation?

* Does 1 degree Celsius make a big difference in transformation efficiency?

* What is selective pressure?

* What is DNA ligation?

* When I keep the cells on ice for 30 minutes, do I keep them litterally on crashed ice or make a water bath which contains crushed ice?
    * Just use crushed ice, you'll risk contamination if water gets around the lid of the test tube.

* Is liquid media or plate culture better for transformation protocols?

* Does agar work for transformation?

* Has anyone done research on using pinapple 'juice' from fresh pineapples to digest bromelain for LB?

* How much PEG-3350 would I need to do a transformation?

* How do I remove agar from LB broth?
    * To get broth instead of agar, just dissolve the appropriate amount of the powder in cold deionised water, let the insoluble agar settle, then decant, pour through coffee filter, and voila. Agar doesn't dissolve until heated so it is easy to remove.

* Can I use an aquarium heater as a waterbath incubator?

* Is it possible to isolate a unknown compound through HPLC?
    * Hi, long story short, yes it is. However, you may not find it as simple as just just injecting a mystery liquid and collecting nice clear fractions of separate compounds. Big questions would be, what solvent to use? what column to use? How do you discern what fractions are "known" compounds and what are "unknown" even if you do get nice fractions? I'd maybe start with a TLC plate, unless you have a MALDI-MS or another LCMS then perhaps you can just start firing away and let the database do the hard work. Even after HPLC, you'd still have to run IR, NMR, MS to then figure out what it is you even got, and if it's new, but, that sounds like a fun challenge to me! Here is a quick return on a google search for screening for new compounds from plant extracts: [1](http://www.wiley-vch.de/books/sample/3527315306_c01.pdf), [2](http://old.iupac.org/symposia/proceedings/phuket97/hostettmann.pdf), [3](http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218439/). Perhaps someone can give you a better, more detailed explanation of the steps to take, good luck, and is this for school or for a personal project?

* When I make an LB Amp Agar Dish, I first mix water and LB Agar. Then I heat it up. I think making 50 ml will be enough for one or two petri dish. Then I have to sterilize it. Will 3 mins in the microwave do the job, or will I need to put it into the autoclaveur for 20mins as recommended?

* How do I make my own custom plasmid vector?

* Would it be feasible for an amateur with access to limited but decent lab supplies to complete a custom plasmid vector?

* How can I make DNA pellets dissolve faster after ethanol precipitation?

* What possible alternatives are there to phenol:chloroform extractions?
    * see <http://groups.google.com/group/diybio/browse_thread/thread/da735df04bfc4067>

* I need to refresh my math/stochiometry skills, what do you recommend for calculating volumes and concentrations?
    * see <http://www.amazon.com/Organic-Chem-Lab-Survival-Manual/dp/0470494379/ref=ntt_at_ep_dpt_1>
    * see <https://groups.google.com/d/msg/diybio/tAqtvhoqT3k/AJBmyTncMgsJ>
        * How do you build a formula from scratch?
        * How do you know which side of the equation to place a known value?
        * How can you check if your answer is in the correct units of measurement?

* What is the meaning of dilutions written like "1:1000"?

* If I have a solution of ampicillin at 1:1000 and I have to add it to 2 mL of LB-solution, how do I do that?

* What is PCR?
    * <http://en.wikipedia.org/wiki/PCR>
    * <http://www.amazon.com/PCR-Basics-M-J-McPherson/dp/0415355478>

* How much is the delay in the PCR machines currently being built for the DIYbio community? How does that compare with commercial lab equipment? Were any design changes made to address this and how much of an affect is this on the reaction?

* How quickly does the OpenPCR change temperature (ramp rate)?
    * OpenPCR is somewhere in the range of 2C/sec. The lightbulb PCR is something like 0.5C upwards and 2-5C downwards.
    * Hot air systems like cyclercan can be as high as 5C/sec.

* What DIY thermocyclers exist?
    * [openpcr](http://openpcr.com) (Josh Perfetto, others)
    * cyclercan
    * [lightbulb pcr](http://russelldurrett.com/lightbulbpcr.html) (Russell Durrett, others)
    * [wiremound-pcr](http://github.com/kanzure/wiremound-pcr) (Stacey Kuznetsov, others)
    * [personalpcr](http://personalpcr.com/) (Chris Templeman, others)
    * coffeecup pcr ([instructables](http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS))
    * manual waterbath

* How do I make a thermocycler?

* How do people go about getting bacteria, plasmids, or other reagents for their experiments?

* Which companies supplies reagents and consumables?

* Will I run into problems as an individual ordering reagents?

* Is this lack of access to reagents a drag on the development of DIYbio?

* What are gene patents?

* Has the human genome been completely sequenced?

* Is it spectrophotometer or photospectrometer?

* Why is the lux operon so useful?

* Are plasmids with the lux-operon available anywhere?

* How do you extract DNA from human spit?

* How do you purify DNA for sequencing?

* How do you resuspend DNA?

* What bioinformatics projects involve the GPU?

* Can you do alignment on a GPU?

* Hey, I always read the term 'Bio Bricks'. Is there any definition? Are a few base-pairs yet Biobricks or does this term begin with plasmid DNA?

* I am a programmer, what should I read to learn about molecular biology?
    * Start here: <http://www.biostat.wisc.edu/~craven/hunter.pdf>

* Why do plates have 96 wells?

* What's a good place to mail some DNA to to get it sequenced; and what process do you use to prepare the sample and to mail it?

* What is a primer?

* How much do primers cost?

* Where can I get an exome sequenced?

* Which math subjects are handy for molecular biology?
    * number theory
    * combinatorics
    * statistical mechanics, see <http://www.youtube.com/watch?v=H1Zbp6__uNw>
    * probability and statistics
    * numerical analysis
    * information theory
    * <http://en.wikipedia.org/wiki/Regular_expression>
    * differential equations
    * linear algebra
    * abstract algebra

* Why are there no really good biology forums?

* Does anyone know how a hobbyist might go about identifying a yeast strain? Is genetic sequencing the only way? If so, where would one send a sample?

* What file format of sequence genomes contains both ORFs and descriptions of what the putative gene functions are?

* What software are available for convenient searching and viewing of ORFs of genomes?

* Okay then, for the time being I only have about £25- £50. What shall I get as an introduction to synth biology?

* What are some good books for biology? <a name="books" />
    * [ck12 biology](http://www.ck12.org/browse/biology/)
    * [General Biology](http://en.wikibooks.org/wiki/General_Biology)

* Where can I get free samples?

* Is there a beginner's guide to synthetic biology?

* Which part of the fruit (peel, pulp, seed) has most DNA and does it change with ripeness?

* What is microfluidics?

* What is NOT microfluidics?

* What's a simple way to make homebrew microfluidic valves?

* Can you use baker's yeast to do cloning?

* How can I make a glowing carrot?

* Is it possible to create my own glowing fruit flies?

* What other introductory or "hello world" projects are there besides glowing broth?

* Is there a phage kit for making cDNA libraries?

* Do scientists really need a PhD?

* Where can I find more detailed information about the MW, structure, and amino acid sequence of lysozyme?

* How does seeking the DNA of a bacterium in an overwhelming quantity of other DNA (blood, feces, etc.) work?

* How do I sterilize a particular culture?

* Is sterilizing a generic process or do I need a different sterilization approach depending on the experiment?

* Is there any danger of killing the experiment if I apply the wrong sterilization method?

* Is there any safety risk for the environment or me during sterilization?

* Should I wear a mouth piece or hair piece while working with DNA? Is this to protect me or to protect my experiment?

* What do I do with biological waste?

* What do I do with electronic waste?

* What material is safe to pour down the drain?

* What is the central dogma of molecular biology?

* What is a good book for an introduction to 454 sequencing or PCR?

* Are there any poly(T) oligo columns on sale that have a free 3' end?
    * <http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Purification-and-Analysis/napamisc/mRNA-Isolation-Dynabeads.html>
    * <http://www.mrcgene.com/oligo1222.htm>
    * <http://www.neb.com/nebecomm/products/productS1560.asp>
    * <http://www.miltenyibiotec.com/en/PG_626_143__MACS_mRNA_Isolation_Kits.aspx>
    * <http://www.isotexdiagnostics.com/mrna_stat-30_kit.html>
    * <http://www.stratagene.com/products/displayProduct.aspx?pid=388>

* When a protocol says to centrifuge 150g for 5min, how was it that they settled on 5min?
    * Is this a feel-good timing parameter based on standard conditions (temperature, etc) where a solution is generally separated?
    * Is there a way to sense this separation with a measurement sensor so that "5 minutes" can be replaced with "until sensor indicates solution is separated"?

* Why do many controllers for gel electrophoresis go to higher voltages (several hundred VDC) if a few volts will suffice- does it provide greater speed, and does resolution suffer as voltage increases?

* When it comes to gel electroimmunodiffusion, are the rules the same as with gel electrophoresis for proteins?

* Is the mechanism by which molecules move  in the gels during electrophoresis fully understood? How do the molecules of different molecular weights, lengths, primary, secondary and tertiary configurations and charges move and get separated?

* What is the basis of using vertical and horizontal gels for the same class of molecules?

* Are there any micro-chemical sets that I can purchase?

* What is the best source for your basic lab setup? (glassware, pH meters n' such)

* What are the advantages and disadvantages of using a pressure cooker versus an autoclave?

* How high does the temperature get inside pressure cookers and autoclaves?

* Are steam sterilizers from tat shops just as good as any other autoclave?

* Why can't I just lyse cells, inactivate RNAse, rid of lipids and protein, extract nucleic acids using alcohol, reverse transcribe using a primer, replicate using complementary-primer, perform blunt-end ligation, and finally transform and plate on antibiotic (assuming vector has resistance)?

* Could a DNA/RNA purification be performed, and then just run through a poly-T column?

* When designing a primer, do you want to begin at the start AUG codon as close to the 5' cap as possible?

* Sambrook says, after the phenol/chloroform, precipitate with alcohol and store in DEPC water or formamide... if I want to go straight to reverse transcription, would I resuspend in TBE?

* If I took said TBE/RNA solution, reverse transcribed, PCRed lightly, and ran on a gel... what do you think the best way to isolate from the gel is? I read about sugars and such inhibiting enzymatic reactions after extraction, so maybe the first step would be to use polyacrylimide gel, then electroelution into a dialysis bag?

* What equipment can I build from scratch to do genetic engineering?

* What cells, dna, proteins, plasmids, protocols, etc... are not patented/copyrighted or can be used in an opensource free way?

* How can I protect my work from being stolen by those who would copyright it and prevent me from using it without their permission or forcing me to pay them to use it?

* How can I force those who use my work to opensource any changes they make if they did use my work?

* There is a tutorial online, for example, on extracting DNA from peas. What steps would I need to take to get that DNA ready for electrophoresis?

* I recently picked up a Pipetman P1000 on eBay. Can anyone recommend tips for it?

* Do only PhDs answer these FAQ questions?

* How do I use a micropipettor?

* How do I calibrate a micropipette?

* What is a good source of pipette tips?

* Are used pipette tips okay to re-use?

* Are tips required to use pipettes?

* How does a pipette or pipettor work?

* Can someone explain to me what problems bioinformatics was created to solve?

* What is necessary to get involved in DIYbio?

* I wonder, though as synbio "grows up" what are some small business ideas that a university student could create with synbio? (on a university student's budget).

* How does one protect intellectual property on a zero budget? Can defensive publishing do the job?

* What are the markets available to small biz synbio?

* How do you make your own calcium chloride?

* So, once an amateur has become acquainted with running a gel, what's next?

* What can I do, in one hour, to make DIYbio better for all of us?

* How can I determine the pH of my municipal water supply?

* What is BLAST?

* What is a lab on a chip?

* Is ethidium bromide staining dangerous to my health?

* Are there any grants for diybio projects?

* Are graduate students welcome in the DIYbio community?

* Will I be put on some (United States) government list for sending emails about DIYbio?

* Who should I contact with practical biosafety concerns?

* How should I dispose of lab materials?

* Is it safe to eat glow-in-the-dark yogurt?

* Is there a way to address the accountability, safety, security concerns of the suppliers and make the more common tools avalable to individuals?

* What safety measures are required for the green gene transformation kit?

* Is it safe for everyone to be biohacking?

* What is DNA?

* What is RNA?

* What is a protein and enzyme?

# Glossary of terms and definitions

* ecoli = escherichia coli; a type of bacteria that you will learn to love
* strain
* bioinformatics
* molecular biology
* synthetic biology
* genetic engineering
* DNA
* RNA
* mRNA
* dsDNA
* NT
* NTP
* dNTP
* pol
* UV
* IR
* microwave
* B. subtilis
* autoclave
* waterbath
* microscope
* FBI
* DIY
* electroporation
* transformation
* electrocompetence
* competence
* log phase
* lag phase
* exponential growth
* midlog
* media
* culture
* plate
* colonies
* GFP
* ampicillin (amp)
* TSS competency
* PEG
* BAP
* LB (lysogeny broth)
* EDTA
* PCR -- polymerase chain reaction
* rtPCR -- real-time polymerase chain reaction
* SNP -- single nucleotide polymorphism
* CSQ -- citizen science quarterly
* LOAC

## A-Z glossary terms: 'A is for Autoclave'

<https://docs.google.com/document/d/10OlwuiGhIIToPlzpywQhOL0hNyAWdZsR06DPubjlcrM/edit>