DNA sequencing: Difference between revisions
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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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==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
* 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/ $
* 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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