DNA sequencing: Difference between revisions
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==Gel Electrophoresis== |
==Gel Electrophoresis== |
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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. |
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[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
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==Supplies Needed== |
==Supplies Needed== |
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⚫ | * 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.] |
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* 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] |
* 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] |
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* 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] |
* 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] |
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* Pipette tips (sterile, for p10 and p200 pipettes) $40 |
* Pipette tips (sterile, for p10 and p200 pipettes) $40 |
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* 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] |
* 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] |
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* 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] |
* 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] |
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* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $ |
* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $25] |
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* 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] |
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* 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] |
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* Pure, DNA-free water $2 (bottled distilled water works) |
* Pure, DNA-free water $2 (bottled distilled water works) |
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⚫ | * 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) |
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