DNA sequencing: Difference between revisions

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DNA barcoding enables the discovery of new species, the determination of which features delineate species, and the building of phylogenetic trees which show how various organisms are related.
DNA barcoding enables the discovery of new species, the determination of which features delineate species, and the building of phylogenetic trees which show how various organisms are related.


A video which shows the whole DNA barcoding process is here: https://www.youtube.com/watch?v=myDjsR_XWhg&t=53s
Step by step photos of the whole process are available here: https://www.facebook.com/groups/FungalSequencing/permalink/2268745516680176/


A video which shows the whole DNA barcoding process is here: https://www.facebook.com/groups/FungalSequencing/permalink/2276807085874019/
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== Location of equipment in CCL ==


How to upload DNA sequences to Genbank: https://www.facebook.com/groups/FungalSequencing/permalink/2163215977233131/
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* The Bio-Rad PCR machine is in the back locked room.
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* There is a 24 well PCR machine near the BioSafety Cabinet.
* Primers and PCR Master Mix are in the -40 freezer.
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* Gel Boxes are in the BSL2 room (in the back right of CCL).
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* Electrophoresis power supplies are on the lab bench nearest the freezers.
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* DNA extraction chemicals, Ethidium Bromide and forceps are in the DNA sequencing cabinet (near the -80 freezer)
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* PCR tubes and pestiles and autoclaved tubes are in the drawers near the -80 freezer.
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* If you can't find something or have questions on anything, email alanQQQQrockefeller at gmail.com, but remove the QQQQ as that was added to foil spam robots.


== DNA Extraction ==
== DNA Extraction ==
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==Supplies Needed==
==Supplies Needed==


* Gel box with tray and comb [http://www.ebay.com/itm/Horizontal-Gel-Electrophoresis-Life-Technologies-Horizon-58-/262519987401?hash=item3d1f691cc9:g:clYAAOSwU1FXP5qS $115] or [http://citizensciencequarterly.com/2011/10/cheapass-science-gel-box/ make your own for $21]
* Gel box with tray and comb [https://www.ebay.com/sch/i.html?_nkw=Gel+Electrophoresis about $100] or [http://citizensciencequarterly.com/2011/10/cheapass-science-gel-box/ make your own for $21]
* PCR machine [http://www.ebay.com/sch/i.html?_from=R40&_trksid=p2380057.m570.l1313.TR1.TRC0.A0.H0.XPCR+machin.TRS0&_nkw=PCR+machine&_sacat=0 $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)
* 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 $27] [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]
* 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 [http://www.ebay.com/itm/PCR-Master-Mix-5x-1-ml-/301021084620?hash=item4616413bcc:g:PTUAAOxyRNJSiugD $46 for 200 reactions] [http://www.the-odin.com/taq-polymerase-master-mix-5x-500ul-50-reactions/ $20 for 50 reactions]
* PCR master mix [https://www.ebay.com/sch/i.html?_nkw=PCR+Master+Mix $50 for 200 reactions] [http://www.the-odin.com/taq-polymerase-master-mix-2x-1ml-40-reactions/ $30 for 40 reactions]
* Electrophoresis DC power supply [http://www.ebay.com/itm/E-C-Apparatus-Corporation-EC-103-Electrophoresis-DC-Power-Supply-Tested-Working-/131885550688?hash=item1eb4fdcc60:g:zSwAAOSw-KFXfVHp $57]
* 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
* 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 $20 for 120 tubes]
* 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]
* Ethidium Bromide or other DNA stain [http://www.the-odin.com/gel-green-like-dna-stain-for-gels-10-000x-100ul/ $10]
* Ethidium Bromide or other DNA stain [http://www.the-odin.com/gel-green-like-dna-stain-for-gels-10-000x-100ul/ $10]
* 100 bp DNA Marker [http://www.the-odin.com/dna-ladder-100bp-100ul/ $10]
* 100 bp DNA Marker [http://www.the-odin.com/dna-ladder-100bp/ $10]
* Agarose [http://www.the-odin.com/agarose-10-grams/ $10]
* Agarose [http://www.the-odin.com/agarose/ $10]
* DNA extraction supplies (NaOH and TRIS buffer, plastic pestles) [http://www.ebay.com/itm/Kodak-sodium-hydroxide-CAT-190-1040-NEW-IN-PACKAGE-BRAND-NEW-/201408293567?hash=item2ee4deaabf:g:t-gAAOSw8cNUOWV4 $10] [http://www.the-odin.com/tris-base/ $10] [http://www.ebay.com/itm/100-Kimble-749521-1590-PELLET-PESTLE-Disposable-Tissue-Grinder-for-1-5mL-Tubes-/331929803679?hash=item4d488edb9f:g:IWQAAOSwyLlXpLL4 $55 for 100 pestles] [http://www.the-odin.com/1.5ml-microcentrifuge-tubes-sterile-500-1000-5000/ 500 tubes for $10]
* DNA extraction supplies (NaOH and TRIS buffer, plastic pestles) [https://www.ebay.com/sch/i.html?_nkw=Sodium+Hydroxide $10] [https://www.ebay.com/sch/i.html?_nkw=TRIS+base $10] [https://www.ebay.com/itm/BEL-ART-Plastic-Disposable-Pestle-3-1-2-8-5cm-Autoclavable-Sterile-70-PACK/332965901495 $45 for 70 pestles] [http://www.the-odin.com/1.5ml-microcentrifuge-tubes-sterile-500-1000-5000/ 1000 tubes for $25]
* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $10]
* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $10]
* Gel loading dye [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5]
* Gel 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]
* 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]
* 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 and orange glasses for viewing DNA on gel [http://www.the-odin.com/trans-eye-luminator-transilluminator-glasses/ $10]
* Blue LED light and orange glasses for viewing DNA on gel
* Pure, DNA-free water $2 (bottled distilled water works)
* Pure, DNA-free water $2 (bottled distilled water works)




* Total cost: $895 (or about $200 less if you DIY some of the pieces)
* Total cost: $895 (or about $200 less if you DIY some of the pieces)


⚫
== Location of equipment in CCL ==

⚫
* The Bio-Rad PCR machine is in the back locked room.
⚫
* There is a 24 well and two 96 well PCR machines near the BioSafety Cabinet.
* Larger stocks of primers and PCR Master Mix are in the -40 freezer. Smaller aliquots of primers and PCR master mix are in the 4 degree fridge.
⚫
* Gel Boxes are in the BSL2 room (in the back right of CCL).
⚫
* Electrophoresis power supplies are on the lab bench nearest the freezers.
⚫
* DNA extraction chemicals, Ethidium Bromide and forceps are in the DNA sequencing cabinet (near the -80 freezer)
⚫
* PCR tubes and pestiles and autoclaved tubes are in the drawers near the -80 freezer.
⚫
* If you can't find something or have questions on anything, email alanQQQQrockefeller at gmail.com, but remove the QQQQ as that was added to foil spam robots.
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