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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.
A video which shows the whole DNA barcoding process is here: https://www.facebook.com/groups/FungalSequencing/permalink/2276807085874019/
== Location of equipment in CCL ==▼
How to upload DNA sequences to Genbank: https://www.facebook.com/groups/FungalSequencing/permalink/2163215977233131/
* The Bio-Rad PCR machine is in the back locked room. ▼
* There is a 24 well PCR machine near the BioSafety Cabinet. ▼
* 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.▼
== DNA Extraction ==
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==Supplies Needed==
* Gel box with tray and comb [
* PCR machine [
* 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 $
* PCR master mix [
* Electrophoresis DC power supply [
* 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 $
* 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
* Agarose [http://www.the-odin.com/agarose
* DNA extraction supplies (NaOH and TRIS buffer, plastic pestles) [
* 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]
* 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 and orange glasses for viewing DNA on gel
* Pure, DNA-free water $2 (bottled distilled water works)
* Total cost: $895 (or about $200 less if you DIY some of the pieces)
▲== Location of equipment in CCL ==
* 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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