Counter Culture Labs

DNA sequencing: Difference between revisions

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== Overview ==
 
DNA barcoding enables the identification of unknown organisms, discovery of new species, the determination of which features delineate species, accurate determination of the range of a species and the building of phylogenetic trees which show how various organisms are related. The most powerful thing that DNA barcoding does is it tells you which collections are the same species. If your goal is to get insights into strains of the same species or learn which genes are present, you'll need to use a different process called full genome sequencing.
 
Step by step photos of the whole process are available here: https://www.facebook.com/groups/FungalSequencing/permalink/2268745516680176/
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Jerry Cooper's excellent Notes on home-based PCR for barcoding fungi: https://www.funnz.org.nz/sites/default/files/DIY%20DNA%20PCR_2.pdf
 
Nanopore DNA sequencing protocol by Stephen Russell: https://www.protocols.io/view/ont-dna-barcoding-fungal-amplicons-w-minion-amp-fl-36wgq7qykvk5/v2/protocols
 
If you would like to send in a mushroom and pay about $25 to have it barcoded, you can send to to http://alvalab.es in Spain.
 
You can also send mushrooms to the Ohio Mushroom DNA Lab for sequencing. They accept donations but do not charge a fee. https://docs.google.com/document/d/1nSKlouFy1Z3OXbCf2cWiKH0hkDnFKZvPn6_oCr3vEK0/edit
 
== DNA Extraction ==
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Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples https://nature.berkeley.edu/garbelotto/downloads/naohextraction2013.pdf
 
In a pinch, plain water can be used as DNA extraction solution.
 
== Polymerase Chain Reaction (PCR) ==
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==Gel Electrophoresis==
 
 
Electrophoresis can tell you if your PCR worked. You can skip this step if you want to save time - you will spend a little more on sequencing PCR reactions that did not work.
 
 
[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis]
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Lower cost sequencing options are available, though you need to mail in the PCR products rather than having them picked up if you aren't near the lab. Two low cost Sanger sequencing options are [https://www.eurofinsgenomics.com/en/products/dna-sequencing/all-sequencing-options/ Eurofins] and [http://www.mclab.com/DNA-Sequencing-Services.html McLab]. Mclab I thinkis the best way to do it is do slightly largerleast reactions - maybe 35 uLexpensive, andat then$5 useper McLabsample for botha forwardbidirectional andread reversewith reads.PCR cleanup (if Ityou costsorder $68 perreactions sampleat a time), $4which is less than justhalf oneof read fromwhat Genewiz charges. Often the forward works and the reverse doesn't, or vice versa - usually one of them works unless you have multiple templates being amplified or some other problem. SlightlyMclab largercan reactionsbe a little tricky to order from because McLabthey requestswant largeryou sampleto sizesfill thanout Genewiz,an andExcel order form - it's not difficult if you needhave toa submittemplate forwardto andwork reversefrom, readsavailable separately[https://images.mushroomobserver.org/Mclab%20DNA%20barcoding%20sample%20form.xlsx here.]
 
 
 
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==Supplies Needed==
 
* PCR machine [https://www.ebay.com/sch/i.html?_nkw=PCR+machine $349] (avoid the cheap Thermo Hybaid brand, Josiah says these break really easily. Good ones are Biorad, MJ Research (usually about $400) and Applied Biosystems (reliable and cheap, like $100, but huge - shipping is like $75)▼
* Gel box with tray and comb [https://www.ebay.com/sch/i.html?_nkw=Gel+Electrophoresis about $100] or [https://cheapassscience.wordpress.com/2011/10/22/how-to-build-a-21-gel-box/ make your own for $21] [https://www.youtube.com/watch?v=sSKls2kNC4U The Thought Emporium on Youtube also has a cheap gel box.]▼
▲* PCR machine [https://www.ebay.com/sch/i.html?_nkw=PCR+machine $349] (avoid the cheap Thermo Hybaid brand, Josiah says these break really easily. Good ones are MJ Research (usually about $400) and Applied Biosystems (reliable and cheap, like $100, but huge - shipping is like $75)
* 10 uL and 200 uL pipettes [http://www.ebay.com/itm/NEW-Oxford-10-50uL-Adjustable-Micro-Pipette-Series-3000-Sampler-System-Pipet-/291224844297?hash=item43ce5a7809:g:lQIAAOSwxCxT96LG 10 - 50 uL $20] [http://www.ebay.com/itm/Brand-New-Single-Channel-Adjustable-Pipetman-20-200uL-p200-pipet-Pipette-j2e-/182032059910?hash=item2a61f4ce06:g:40QAAOSwiylW~eU2 P200 $22]
* PCR master mix [https://www.the-odin.com/taq-polymerase-master-mix-2x-1ml-40-reactions/ $30 for 40 reactions] [https://empiricalbioscience.com/shop/rae/grouping-2-rae/taq-2x-colorless-mastermix-4/ $88 for 500 reactions]
* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50]▼
* Pipette tips (sterile, for p10 and p200 pipettes) $40
* PCR tubes [http://www.ebay.com/itm/Azzota-PCR-Tubes-Flat-8-strip-Caps-120pcs-pk-/252184303378?hash=item3ab75b1712:g:9DUAAOSwt6ZWVJCA $10 for 120 tubes]
* DNA stain [http://www.the-odin.com/gel-green-like-dna-stain-for-gels-10-000x-100ul/ $10]▼
* 100 bp DNA Marker (optional) [http://www.the-odin.com/dna-ladder-100bp/ $10]▼
* Agarose [http://www.the-odin.com/agarose/ $10]▼
* DNA extraction supplies (or IBI X-Amp) (NaOH and TRIS buffer, optionally plastic pestles. If you get pestles get the size for the .5 ml tubes so you can use 200 uL PCR tubes for DNA extractions) [https://www.ebay.com/sch/i.html?_nkw=Sodium+Hydroxide $10] [https://www.ebay.com/sch/i.html?_nkw=TRIS+base $10]
* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $1025]
* Gel loading dye (not necessary if you get master mix that already has loading dye) [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5]▼
* TAE buffer concentrate [http://www.the-odin.com/tae-buffer-mix-10-l-50g/ $10]
* Tube rack for DNA extracts [http://www.the-odin.com/microcentrifuge-tube-rack-60-tube-2-sided-1-5-ml-and-0-5-or-0-2-ml-reversible/ $5]
* Blue LED light (490 nanometer) and orange glasses for viewing DNA on gel (a 365 nanometer UV flashlight works even better - no orange glasses needed) [https://www.amazon.com/dp/B005IPPBNI $17]▼
* Pure, DNA-free water $2 (bottled distilled water works)
▲* Gel box with tray and comb [https://www.ebay.com/sch/i.html?_nkw=Gel+Electrophoresis about $100] or [https://cheapassscience.wordpress.com/2011/10/22/how-to-build-a-21-gel-box/ make your own for $21] [https://www.youtube.com/watch?v=sSKls2kNC4U The Thought Emporium on Youtube also has a cheap gel box.] (optional)
▲* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50] (optional)
▲* DNA stain [http://www.the-odin.com/gel-green-like-dna-stain-for-gels-10-000x-100ul/ $10] (optional)
▲* 100 bp DNA Marker (optional) [http://www.the-odin.com/dna-ladder-100bp/ $10]
▲* Gel loading dye (not necessary if you get master mix that already has loading dye) [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5] (optional)
▲* Blue LED light (490 nanometer) and orange glasses for viewing DNA on gel (a 365 nanometer UV flashlight works even better - no orange glasses needed) [https://www.amazon.com/dp/B005IPPBNI $17] (optional)
▲* Agarose [http://www.the-odin.com/agarose/ $10] (optional)
 
 
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