Editing DNA sequencing

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A video which shows the whole DNA barcoding process is here: https://www.facebook.com/groups/FungalSequencing/permalink/2276807085874019/
 
A video which shows the whole DNA barcoding process is here: https://www.facebook.com/groups/FungalSequencing/permalink/2276807085874019/
 
YouTube playlist with DNA barcoding videos: https://www.youtube.com/playlist?list=PLi7dEmBMB3xvyt1OY7l18BXR1VpLZcLsP
 
 
Sigrid's PCR protocol: https://docs.google.com/document/d/13B9OSE_ar_vWWZnHZegr2FROnMak78qHZxEZXc1E9jk
 
   
 
How to upload DNA sequences to Genbank: https://www.facebook.com/groups/FungalSequencing/permalink/2163215977233131/
 
How to upload DNA sequences to Genbank: https://www.facebook.com/groups/FungalSequencing/permalink/2163215977233131/
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[[http://i.imgur.com/9fLgFqJ.jpg|thumb|left|alt=NaOH DNA Extraction]]
 
[[http://i.imgur.com/9fLgFqJ.jpg|thumb|left|alt=NaOH DNA Extraction]]
   
Another good way to extract DNA is with IBI X-Amp. PCR success is as good or better than the other methods, and there's not as much that can be messed up since you don't need to mix any chemicals. https://www.ibisci.com/products/x-amp-dna-reagent
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Another good way to extract DNA is with IBI X-Amp. PCR success is as good or better than the other methods, and there's not as much that can be messed up since you don't need to mix any chemicals. [https://www.ibisci.com/products/x-amp-dna-reagent<nowiki>]</nowiki>
 
   
   
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I use a 25 μL [https://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] reaction which includes 1 μL of 10 μM forward [https://en.wikipedia.org/wiki/Primer_(molecular_biology) primer], 1 μL of 10 μM reverse primer, 1 μL DNA template and the [https://www.google.com/search?q=pcr+master+mix&oq=PCR+master+mix PCR master mix]. I use the Taq Meangreen 2x Master Mix [https://empiricalbioscience.com/shop/rae/grouping-3-rae/taq-2x-meangreen-master-mix-copy-2/<nowiki>], which already has loading dye included.</nowiki>
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I use a 25 μL [https://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] reaction which includes 1 μL of 10 μM forward [https://en.wikipedia.org/wiki/Primer_(molecular_biology) primer], 1 μL of 10 μM reverse primer, 1 μL DNA template and the [https://www.google.com/search?q=pcr+master+mix&oq=PCR+master+mix PCR master mix]. I use the Taq Keengreen 2x Master Mix [https://www.ibisci.com/products/ibi-taq-keengreen-2x-master-mix?_pos=1&_sid=46411e7d6&_ss=r<nowiki>], which already has loading dye included.</nowiki>
   
 
The current PCR program I am using is an initial denaturation of two minutes at 95 degrees C, followed by 30 cycles of denaturation at 95 degrees for 30 seconds, annealing at 54 degrees for 30 seconds and an extension phase of 72 degrees for 55 seconds. Since the DNA we are amplifying typically isn't very long, it is probably ok to omit the final extension phase of 6 minutes at 70 degrees. The 54 degree annealing temperature was chosen by looking up the [http://biotools.nubic.northwestern.edu/OligoCalc.html melting point] of the primers and subtracting 3 - 5 degrees.
 
The current PCR program I am using is an initial denaturation of two minutes at 95 degrees C, followed by 30 cycles of denaturation at 95 degrees for 30 seconds, annealing at 54 degrees for 30 seconds and an extension phase of 72 degrees for 55 seconds. Since the DNA we are amplifying typically isn't very long, it is probably ok to omit the final extension phase of 6 minutes at 70 degrees. The 54 degree annealing temperature was chosen by looking up the [http://biotools.nubic.northwestern.edu/OligoCalc.html melting point] of the primers and subtracting 3 - 5 degrees.
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==Supplies Needed==
 
==Supplies Needed==
   
* 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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* 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 [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)
 
* 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]
 
* 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]
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* PCR master mix [https://www.the-odin.com/taq-polymerase-master-mix-2x-1ml-40-reactions/ $30 for 40 reactions]
 
* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50]
 
* 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
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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]
 
* 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 (490 nanometer is best) and orange glasses for viewing DNA on gel [https://www.amazon.com/dp/B005IPPBNI $17]
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* 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)
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== Location of equipment in CCL ==
 
== Location of equipment in CCL ==
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