|
Some of these posts are very old and might contain outdated information. You may wish to search for newer posts instead.
|
ebass
Stranger


Registered: 03/15/06
Posts: 387
Last seen: 11 years, 1 month
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5586798 - 05/03/06 05:27 PM (17 years, 8 months ago) |
|
|
isn't genetic drift due to chance events?
|
texas34
Lurker

Registered: 12/03/05
Posts: 23
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
#5588484 - 05/03/06 11:51 PM (17 years, 8 months ago) |
|
|
Well yes, but genetic drift breaks down as the effective population size decreases. That is alleles are fixed or lost in the population even without selective pressure. In fact, even alleles under positive selection may be lost if the effective population size becomes too small. I image the strains from distant locations are in separate populations and would have likely fixed as many different alleles as the same. In addition, mutations within each population are likely to be unique.
|
BlimeyGrimey
Collector of Spores



Registered: 08/24/05
Posts: 3,788
Loc: Puget Sound
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
#5589315 - 05/04/06 08:43 AM (17 years, 8 months ago) |
|
|
Quote:
Feelers said: When we did it it was just with a blue dye - no flourescent stuff, and it worked ~ok. But yeah that would definately be an improvement.
yea , i'll be using 'Azure A' blue staining. alot safer than all the mutagenic flourescent dyes, and needs no UV lighting to see the dna. save and flatbed scannable. yes mycogirl, this dye works without UV light, its the same setup they use in high school biology classes because its safe, however it dyes the whole gel block blue (see-thru blue) but the dna fragments show up a deep dark blue.
and fastfred has the right idea about what i'm doing. its not to setup a Cube Genome, that would take years and alot of unbuyable equipment. i'm trying to show the variation between strains to see if some of the "strains" people buy are actually the same strain or not.
i'll be using spores as a source of dna for a few reasons. A. its legal (vs illegal shrooms and mycelium) so if i decide to give out the results outside of the Shroomery i wont be hassled. B. it'll give a big range of that strain's dna while a mushroom or mycelium would be a limited gene pool. C. it IS possible to use spores using a Qiagen DNeasy Plant DNA extraction kit, which produces 95% and higher pure dna with 5% protein contamination. well within the limits we need.
i will be trying to use a 5kb ladder however all i've been able to find for sale is a 1kb ladder. the gel will have only 6 wells in it. so i'll have ladders on both sides and use 4 strains/samples per gel. not sure what restriction enzyme i'm going to use, but i want to put alot of research into this before i start the project. look up the ncbe , thats where i'm getting the supplies from. NCBE B.
|
texas34
Lurker

Registered: 12/03/05
Posts: 23
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
#5589498 - 05/04/06 10:20 AM (17 years, 8 months ago) |
|
|
Given that you will be digesting the DNA, the 1KB ladder should be fine. In general, you should use two restriction enzymes. One should be a "rare" cutter and the other a more frequent cutter. With only 6 lanes, I wouldn't bother with two lanes of ladder, but that's just me.
|
fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
#5593123 - 05/05/06 07:47 AM (17 years, 8 months ago) |
|
|
If you use two restriction enzymes how do you interpret the results? You'll not really be able to know if you have the same or different restriction sites on both ends of your fragments. I'm not much of an expert on RFLP, so maybe you can clue me in.
As for selecting the restriction enzyme... I would look at some papers and see what is most commonly used for fungal RFLP.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
|
texas34
Lurker

Registered: 12/03/05
Posts: 23
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5593753 - 05/05/06 11:43 AM (17 years, 8 months ago) |
|
|
It doesn't matter what the sequence is at the ends of the fragments. What you are doing with RFLP is looking for the pattern of different size bands on your gel. You will get the same pattern over and over for one isolate (unless a mutation has occurred in it). When comparing more than one isolate/strains/species you are comparing the patterns between them. If you did want to clone a specific band, you just blunt the ends or use blunt end cutters to begin with.
You use two enzymes to ensure that you will get fragments of DNA that are of a size that will separate easily on your gel. Even when using a frequent cutter, you are likely to get some very large fragments that "hang out" together even though they are different in sequence. By adding a rare cutter, you are hoping that some of these very large fragments will contain a site for the rare cutter to give you two smaller fragments. You can look at it the opposite way as well (rare cutter leaves large fragments and frequent cutter chops these up).
For the most part it doesn't matter what enzymes you use as long as you use the same ones for all your digests. However, some are sensitive to things like methylation of DNA bases and the such, so I would make sure to get ones that always cut no matter what.
Edited by texas34 (05/05/06 02:28 PM)
|
Gio
Chelator

Registered: 05/06/06
Posts: 14
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
#5599935 - 05/07/06 02:04 AM (17 years, 8 months ago) |
|
|
"For the most part it doesn't matter what enzymes you use as long as you use the same ones for all your digests. However, some are sensitive to things like methylation of DNA bases and the such, so I would make sure to get ones that always cut no matter what."
|
texas34
Lurker

Registered: 12/03/05
Posts: 23
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5604546 - 05/08/06 11:03 AM (17 years, 8 months ago) |
|
|
I wanted to add another reason to use two restriction endonucleases. With RFLP, only mutations that create or destroy restriction sites are detected. These mutations are detected as an increase or decrease in the number of bands between samples on a gel. By using two enzymes, the number of detectable loci in the genome is increased because the recognition sequence of the two enzymes are different. T34
|
fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
#5631263 - 05/15/06 02:55 AM (17 years, 8 months ago) |
|
|
If you run two different enzymes in separate lanes and then another with both enzymes(a double digest) you can do a restriction site map. That would be pretty cool. I don't know if it would be all that useful, but it's not all that difficult.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
|
No_Life_G33k
Now with 10%less noobness


Registered: 03/08/05
Posts: 356
Last seen: 13 years, 10 months
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5915383 - 07/30/06 03:56 PM (17 years, 6 months ago) |
|
|
Soooooooo...whatever happened to the project???
I'd be willing to throw a some cash in the pot to help get this started.... it'b be nice to see some quality research replace geusswork.
The kits run less then $200 for the kits I've seen on the science supply sites.
Anyone else interested??
Perhaps some underwriting from a sponsor???
BTW- anyone know what shroom genome testing would cost??? The have cheap DNA tests available as mail in tests, so I was wondering if we might be able to do something similar.
|
cloudtop
Stranger


Registered: 08/16/04
Posts: 66
Loc: bespin
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
#5916813 - 07/30/06 10:24 PM (17 years, 6 months ago) |
|
|
I'd be surprised if 25% genetic variation between strains turned up.
Be sure to control both against a single strain twice and against some other Psilocybe just for fun.
-------------------- peacefromabovecloudtop
|
fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: cloudtop]
#5917715 - 07/31/06 04:22 AM (17 years, 6 months ago) |
|
|
> Anyone else interested??
I'm interested. I thought this was a great idea.
With as cheap as it is to do these tests we could pool our funds and hopefully the sponsors would also donate to the project.
As you do more tests it rapidly becomes cheaper. I would bet that all the strains could be done for ~$300. Vendors surely make more than that each day, so you would think they would be interested in something like this.
I've got a lab available to me so the equipment is no problem. I don't work in a "multi-million dollar" lab though, so reagents are ordered on a per-project basis. So I can't just pilfer the supplies.
I have some spare soil DNA extraction kits, but those are for bacteria and I'm not sure that they would work well.
Anyway I'm in for a ten-spot at least. Maybe we could start a petition or try to get the vendors to see the good publicity that would come from doing some good science like this.
-FF
|
Feelers
Anti-Myth-Rhythm-Rock-Shocker


Registered: 06/18/02
Posts: 1,806
Loc: Land of Oz
Last seen: 5 years, 8 months
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5917791 - 07/31/06 06:04 AM (17 years, 6 months ago) |
|
|
Remember, its probably not in many vendors intrests to confirm that all the strains of P. cubensis are genetically identical. 
I suppose it's more of an acedemic interest - I dont think anyone really cares if they are the same - as they are all the same price. Plus you get to give them funny names when you find a weird "strain".
Is hybrid vigor appreciable when say crossing an asian cube with a mexican one? That could be one use of the genetic diversity we are searching for, if it turns up haha!
|
fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
#5917939 - 07/31/06 08:19 AM (17 years, 6 months ago) |
|
|
I don't think that an RFLP would show them to be identical. Actually if there is any truth whatsoever to the origins of the various strains then there should be plenty of differences.
Strains of the same species collected from different locations will usually show several length polymorphisms. It would be very helpful for showing which ones are the same and which ones are different though. And it should be able so show approximate relatedness between strains.
In the end it's just going to produce some gels that very few people here would be able to interpret. BUT it will generate a lot of discussion and debate on the issue and vendors can associate themselves with supporting the cause and being high-tek.
It would be like those Gatorade commercials where they have this athlete hooked up to a dozen electrodes, a breathing tube, etc. And a scientist with a clipboard is standing there pretending to record data. It's complete bullshit. Gatorade was formulated in 1965. They don't really even pretend to be actually doing anything real with all that crap, they just want to associate themselves with high tek. I laugh at that commercial every time I see it.
With RFLP's some good science would actually be getting done, so it would be a lot better than some phoney-baloney crap. It would make for a cool advertisement to have a vendor holding up a gel and pretending to be all scientific. Very good press.
-FF
|
TygerClaw
Stranger
Registered: 12/06/05
Posts: 6
Last seen: 15 years, 3 months
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
#5918043 - 07/31/06 09:22 AM (17 years, 5 months ago) |
|
|
I like this idea, but honestly I don't think it will be as simple as shake n bake, like it sounds. I can see a few technical problems that can/will/always crop up with something like this ^_^ 1. Your using spores, how are you going to get the DNA out? I'm thinking it will be much tougher than doing it with soft mycelium tissue. The kits used in high school use bacteria which is easy as pie to crack open to get the DNA. 2. The DNA your trying to get at is genomic DNA, .. easier to break/shear than the plasmids used in the high school kit. (Plasmids- short ~1-10kb sized normally.. genomic- if stretched straight end to end human genome could reach the moon and back.) 3. Those blue dye kits are very very 'fuzzy' when resolving bands on agarose. Ethidium bromide is better but as someone said its mutagenic and the UV is not user friendly (heh yeah been sunburned by those lamps.. sometimes bein the whitest white boy known has draw backs ^_^) 4. I'm just guessing that the software to really analyze RFLP's is insanely expensive, but there might be a freebie on the net but I would be cautious with it. Course that depends on how 'different' the pattern is/isn't between the strains (and I kinda doubt there'd be a large difference, though that might depend on the enzymes used)
Now these are far from insurmountable problems. ^_^ just things to watch out for when trying to do it. If I keep looking i'm sure I could find other hurdles, but I'm not going to rain on parades here I think its a great idea to do
|
fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: TygerClaw]
#5920985 - 08/01/06 03:58 AM (17 years, 5 months ago) |
|
|
Not quite as simple as shake n bake, but it's certainly a routine procedure done many, many times with plenty of published literature to go from.
1. There are DNA extraction kits that will work well with spores. Live tissue would probably work better, but there are legal problems involved with that. It will be much easier to publish results if those legal issues are avoided.
2. That's why you cut it with a restriction enzyme. Separating genome sized pieces of DNA takes a pulse field electrophoresis unit which is quite spendy and also would eliminate the intended goal of RFLP... Restriction Fragment Length Polymorphisms.
3. The blue dye your talking about is probably the loading buffer. That's just there to make sure you don't run your bands right off the end of the gel. I haven't used the blue dye they are selling nowadays, but I hear it works alright. Ethidium bromide works fine and anyone doing gel electrophoresis should have some on hand. The UV is only a problem if you are foolish and stare at your gel on the transilluminator for too long. You should just be taking a picture of it and not staring at it. The blue dye eliminates those problems though. Getting clear bands is more about technique and parameters than the staining agent. In your class they probably wanted to get it done in one lab period so they ran the gel at too high of a voltage, resulting in the fuzzy bands.
4. No software required. There is software that can do some fancy things, but for our purposes working it out by hand will be just fine.
And go ahead and look for problems. Don't worry about raining on parades. It's better to anticipate potential problems before rather than when you're having them.
The biggest difficulty is going to be in deciding the best restriction enzyme (or combo) to use. Then again it's hard to go wrong there and I'm sure the literature will provide some good clues as to the best one(s) to use. Another potential problem is making sure proper amounts and times are used to get complete digestion of the DNA. Trivial problems for someone who does this stuff all the time, or at least has done a few.
-FF
|
No_Life_G33k
Now with 10%less noobness


Registered: 03/08/05
Posts: 356
Last seen: 13 years, 10 months
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
#5937597 - 08/06/06 04:32 PM (17 years, 5 months ago) |
|
|
Quote:
Feelers said: Remember, its probably not in many vendors intrests to confirm that all the strains of P. cubensis are genetically identical. 
....
I was hoping it would appeal to a truly dedicated vendor, not some schlocky spore-jockey. 
There are some vendors that seem to have their hearts in the hobby.
|
SpookerShroom
Shroomerite
Registered: 06/12/06
Posts: 244
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
#5938250 - 08/06/06 08:29 PM (17 years, 5 months ago) |
|
|
True, I think most vendors would be all in.
If you need any special supplies or more people to start doing labwork let me know, I am in the biotechnology sector and have pretty extensive lab access. I actually was excited when I saw this thread, I've been pondering doing something like this in my spare time.
|
No_Life_G33k
Now with 10%less noobness


Registered: 03/08/05
Posts: 356
Last seen: 13 years, 10 months
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: SpookerShroom]
#5959443 - 08/13/06 02:43 PM (17 years, 5 months ago) |
|
|
So we need someone to do the work, and someone to put up the funding.
Anyone think that FMRC might be interested???
|
Herbus
...

Registered: 10/19/04
Posts: 1,477
Loc: Reading the map...
Last seen: 10 years, 23 days
|
Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
#5965718 - 08/15/06 10:58 AM (17 years, 5 months ago) |
|
|
If nobody has mentioned it, or filled in the blank spots for 'strains' to examine the genetic make-up of, I'd suggest EQ.
Everyone knows how dependable, vigorous and resilient this strain is... It may be useful to compare this strain with others...
-------------------- ...
|
|