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chibiabos
Cosmic Pond Scum



Registered: 03/16/17
Posts: 4,180
Last seen: 10 months, 11 days
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: Alan Rockefeller]
#24723265 - 10/19/17 09:06 PM (6 years, 3 months ago) |
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Why would anybody skip that step? Seems like a pretty big fuckup, with respect to quality control.
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Alan Rockefeller
Mycologist

Registered: 03/10/07
Posts: 48,276
Last seen: 6 hours, 55 minutes
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
#24723274 - 10/19/17 09:09 PM (6 years, 3 months ago) |
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Quote:
chibiabos said: Why would anybody skip that step? Seems like a pretty big fuckup, with respect to quality control. 
Because if you are sequencing 96 reactions at a time and doing your own PCR cleanup, it can cost as little as $2 for Sanger sequencing. For that kind of money, it does not make sense to waste time with electrophoresis because you are only saving $2 per PCR that does not work. Also sometimes there is enough amplified DNA there to get a good sequence, but not enough to show up on the gel. A lot of professionals just sequence everything, and if the sequence does not come back clean they run the PCR again.
For many researchers, the time spent with electrophoresis does not justify saving a couple dollars of their bosses money.
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chibiabos
Cosmic Pond Scum



Registered: 03/16/17
Posts: 4,180
Last seen: 10 months, 11 days
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: Alan Rockefeller]
#24723301 - 10/19/17 09:19 PM (6 years, 3 months ago) |
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Granted, but if you skip that step in the interest of saving money then you still need some way to verify that you were actually studying one sequence instead of several of them. You can always compare it to other sequences in order to justify a guess that it is some particular region of a genome, but unless the way in which you actually end up solving the sequence of your PCR products allows some way to tell how many, distinct, sequences you are solving (without knowing beforehand) then you're making assumptions about your PCR products which you can't really justify. That might be fine in industry, but it's not science.
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Alan Rockefeller
Mycologist

Registered: 03/10/07
Posts: 48,276
Last seen: 6 hours, 55 minutes
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
#24723327 - 10/19/17 09:36 PM (6 years, 3 months ago) |
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Quote:
chibiabos said: Granted, but if you skip that step in the interest of saving money then you still need some way to verify that you were actually studying one sequence instead of several of them.
If you have multiple templates being amplified by the PCR reaction, when you get the chromatogram back it is garbage because you have lots of overlapping peaks. The base caller finds N's all throughout the sequence, it isn't anything like a clean sequence you are supposed to get back.
About 1% of my sequences come back this way, either because there was a fungus eating the mushroom or a pipetting error where I put two PCR products into one tube.
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Stonehenge
Alt Center


Registered: 06/20/04
Posts: 14,850
Loc: S.E.
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
#24725501 - 10/20/17 06:50 PM (6 years, 3 months ago) |
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Chibiabos, you seem very knowledgable in many areas. May I ask what kind of work you do?
-------------------- “A democracy cannot exist as a permanent form of government. It can only exist until the voters discover that they can vote themselves largesse from the public treasury. From that moment on, the majority always votes for the candidates promising the most benefits from the public treasury with the result that a democracy always collapses over loose fiscal policy, always followed by a dictatorship.” (attributed to Alexis de Tocqueville political philosopher Circa 1835) Trade list http://www.shroomery.org/forums/showflat.php/Number/18047755
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