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InvisibleBlimeyGrimey
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The Shroomery Strain DNA-Length Analysis Project!
    #5583452 - 05/02/06 09:53 PM (17 years, 10 months ago)

i'm planning on ordering a home Electrophoresis kit.
the kit will include enough supplies to perform 16 dna tests.

now here's the idea.
test 8-10 common strains (the other tests will be used to calibrate the results) and see what genetic variation there is. maybe this way we could find the truth behind the "re-named" strains. as most know, there is a rumor that the Amazonian strain and PESA strains and others are the same strain. this way we could see if these strains are the same or not. also we can see how big of a difference the 2 red spore strains have vs "normal" strains.

we could also test the PR genetics against the 50% redboy and the 75% redboy etc to see if the PR genetics are really being bred out.

the gel slabs (after staining) could be set on a flatbed scanner and scanned, then each pic could be tinted a different color using a photo editor. this way we could overlay different strain results over eachother to view the similarities/differences.

here's a list of the strains i have in mind to test.

1. PF-C
2. PF-A
3. PF-R
4. Puerto Rican
5. Redboy/PR
6. Penis Envy
7. Malabar
8. (Suggestion needed)
9. (Suggestion needed)
10. (Suggestion needed)

i'd need ALOT of spores for each test.
an easy way to do this would be to use a single dark print and make 2ml of soultion with it.

i'd need these donated as i only have 3 of the listed strains to test.

all donators will be listed (unless they request not to be) in the final report.
i think this would be a valuable addition to the shroomery archives.
any suggestions and donations are much appreciated.

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
    #5583713 - 05/02/06 10:31 PM (17 years, 10 months ago)

Awesome plan BlimeyGrimey!

This is really important work that is overdue. By "DNA-Length Analysis" I assume you mean restriction fragment length polymorphism (RFLP). As far as I know you would be the first to use it on the species you mention. I don't know what sort of DNA purification you plan to do, but I would suggest using mycelium instead of spores. A couple of tips and common mistakes to avoid are: Forgetting to include your ladder, running the DNA off your gel, and using too high of a voltage. I would load each strain's DNA into lanes on the same gel with a ladder on each side. Generally the lower the voltage you use the cleaner your bands. Remember those gels are slippery bastards, so try not to let them end up on the floor. Your choice of restriction enzyme will lock future people into that enzyme if they want to compare data. It might be worthwhile to spend a little time choosing one, but there aren't that many common choices. I'm not sure how you would get a computer scanner to scan well with the UV illumination you'll probably need. It might work better to take a picture and then scan that.

I didn't know they made home electrophoresis kits, that's new to me! It would be great if you could get it done. Good luck!

-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

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InvisibleZen Peddler
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
    #5583735 - 05/02/06 10:33 PM (17 years, 10 months ago)

I look forward to the results but Im not actually sure it will work out that easily.
Wouldnt it be more interesting to test those strains that have been suggested are contentious - rather than chucking in an obviously diverse mixture (Puerto Rican, etc.)

I would eagerly be interested if someone did proper mapping of all of the cubensis strains on the market...


--------------------

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OfflineFeelers
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Zen Peddler]
    #5583906 - 05/02/06 10:55 PM (17 years, 10 months ago)

The beginnings of the Cubensis genome project. :laugh:

Yeah man I've done some of those gel electrophresis things - They arent actually very complicated from what I remember. (we did them at high school)
There are lots of simple things that can go wrong though
Use something to make the wells as uniform as possible (theres a comb in the kit hopefully).
I cant really remember but arent they pretty cheap to do? Whats the gel made of?

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Invisiblemycogirl
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5584086 - 05/02/06 11:32 PM (17 years, 10 months ago)

agarose. (And gel red or ethidium bromide) I thought you always needed a uv light to see the laddering. You also need to make a buffer solution to suspend the agarose and gel red into, and to run the gel on.


--------------------

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5584095 - 05/02/06 11:34 PM (17 years, 10 months ago)

I didn't write this, but here's a detailed explanation:

There are two basic types of materials used to make gels: agarose and polyacrylamide. Agarose is a natural colloid extracted from sea weed. It is very fragile and easily destroyed by handling. Agarose gels have very large "pore" size and are used primarily to separate very large molecules wiht a molecular mass greater than 200 kdal. Agarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior. That is, the bands formed in the agarose gels are fuzzy and spread far apart. This is a result of pore size and it cannot be controlled.
Agarose is a linear polysaccharide (average molecular mas about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%.

Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel.


The polyacrylamide gel electrophoresis (PAGE) technique was introduced by Raymond and Weintraub (1959). Polyacrylamide is the same material that is used for skin electrodes and in soft contact lenses. Polyacrylamide gel may be prepared so as to provide a wide variety of electrophoretic conditions. The pore size fo the gel may be varied to produce different molecular seiving effects for separating proteins of different sizes. In this way, the percentage of polyacrylmide can be controlled in a given gel. By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2,000 kdal. This is the ideal range for gene sequencing, protein, polypeptide, and enzyme analysis. Polyacrylamide gels can be cast in a single percentage or with varying gradients. Gradient gels provide continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. Because of this banding effect, detailed genetic and molecular analysis can be performed on gradient polyacrylamide gels. Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels.

From:
http://www.bergen.org/AAST/Projects/Gel/


-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

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Invisiblemycogirl
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584117 - 05/02/06 11:38 PM (17 years, 10 months ago)

In engligh-

You use agarose for DNA and page or polyacrylamide or western blots for protein analysis.  (These need different apparatus for current conduction as well, one runs horizontally, and one runs vertically)

:smile:


--------------------

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584119 - 05/02/06 11:38 PM (17 years, 10 months ago)

Also you need to add ethidium bromide, which is the fluorescent dye used for staining the DNA. The ethidium bromide then lights up when you put the gel on the transilluminator (UV light box).

I just decided to add that the ethidium bromide goes in the gel, not the DNA as you might expect.

Here is another tidbit from the same source:

To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.

From:
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html


-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

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OfflineFeelers
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5584155 - 05/02/06 11:46 PM (17 years, 10 months ago)

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.

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Invisiblemycogirl
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584161 - 05/02/06 11:47 PM (17 years, 10 months ago)

A word of caution. If you are indeed working with ethidium bromide opposed to Gel Red, please be extremely cautious as it is highly mutagenic. Look up the molecule and note its smooth planar conformation allowing it to insert itself into your DNA and cause it to replicate incorrectly.


--------------------

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Invisiblemycogirl
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5584176 - 05/02/06 11:51 PM (17 years, 10 months ago)

Quote:

Feelers said:
When we did it it was just with a blue dye - no fluorescent stuff, and it worked ~ok. But yeah that would definitely be an improvement.




The sample loading buffer has a blue dye to it so you can see where the DNA is so it doesn't fall off the gel. I would bet that someone took the gel to be analyzed under uv light and you received a picture with white bands on a black background. Or perhaps, they told you where all the bands were. If you could see DNA with a dye, everyone would use that.


--------------------

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Offlineebass
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
    #5584501 - 05/03/06 04:38 AM (17 years, 10 months ago)

Quote:

BlimeyGrimey said:
i'm planning on ordering a home Electrophoresis kit.
the kit will include enough supplies to perform 16 dna tests.

now here's the idea.
test 8-10 common strains (the other tests will be used to calibrate the results) and see what genetic variation there is. maybe this way we could find the truth behind the "re-named" strains. as most know, there is a rumor that the Amazonian strain and PESA strains and others are the same strain. this way we could see if these strains are the same or not. also we can see how big of a difference the 2 red spore strains have vs "normal" strains.

we could also test the PR genetics against the 50% redboy and the 75% redboy etc to see if the PR genetics are really being bred out.

the gel slabs (after staining) could be set on a flatbed scanner and scanned, then each pic could be tinted a different color using a photo editor. this way we could overlay different strain results over eachother to view the similarities/differences.

here's a list of the strains i have in mind to test.

1. PF-C
2. PF-A
3. PF-R
4. Puerto Rican
5. Redboy/PR
6. Penis Envy
7. Malabar
8. (Suggestion needed)
9. (Suggestion needed)
10. (Suggestion needed)

i'd need ALOT of spores for each test.
an easy way to do this would be to use a single dark print and make 2ml of soultion with it.

i'd need these donated as i only have 3 of the listed strains to test.

all donators will be listed (unless they request not to be) in the final report.
i think this would be a valuable addition to the shroomery archives.
any suggestions and donations are much appreciated.


.

So...do we know any markers or are you going to look for them. It will take alot more than 12 tests to figure that out.

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OfflineFeelers
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5584508 - 05/03/06 04:46 AM (17 years, 10 months ago)

Quote:


The sample loading buffer has a blue dye to it so you can see where the DNA is so it doesn't fall off the gel. I would bet that someone took the gel to be analyzed under uv light and you received a picture with white bands on a black background. Or perhaps, they told you where all the bands were. If you could see DNA with a dye, everyone would use that.



You're probably right, it was four years ago - I vaguely remember him taking them away and perhaps the results we got were just printed off.

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5584519 - 05/03/06 04:58 AM (17 years, 10 months ago)

Quote:

ebass said:
So...do we know any markers or are you going to look for them. It will take alot more than 12 tests to figure that out.




I know some good markers... Ultra fine point Sanford Sharpies work good for writing on petri dishes, and they are the de facto standard for lab work. I like the ultra fine point ones, but they don't tend to last as long or write as easily as the fine point ones. Usually it doesn't take 12 tests to figure out if your marker works or if you need a new one. I just try to write a few strokes and if it doesn't work I get a new one. It's not worth it to lick it or fool around with it because it'll just quit writing again in a little bit.

What made you think that it would take more than 12 tests to figure out if a marker works? And what made you ask about markers anyway? And why did you have to quote an entire 25+ line post just to type a one line question that's off topic? Did you get your threads mixed up? Nobody mentioned anything about markers or the number 12...


-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

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Offlineebass
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584554 - 05/03/06 05:32 AM (17 years, 10 months ago)

O.K. then. The number is 16 and sorry for being rude. Question is do you have phenotypes matched with genetic markers?

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5584586 - 05/03/06 06:19 AM (17 years, 10 months ago)

The question is not applicable to the context of this thread. We aren't talking about phenotypes or genetic markers. Associating genotype (or markers) with phenotype is way, way beyond the scope of what we're discussing. That would take millions of dollars and years of research to accomplish anything useful.

In fact, we're not even talking about 18S sequencing that would allow us to construct a phylogenetic tree. (just ignore that sentence)

What Blimey is talking about is RFLP. That is an old, cheap, and easy to do technique. You purify the DNA of an organism. Then you use a restriction enzyme to cut the DNA into bits at sequences specific to the restriction enzyme you use. Then you place the DNA into a gel. You run electricity through the gel. DNA is negatively charged so it migrates through the gel from the anode(-) towards the cathode(+). The DNA has to pass through the gel so longer fragments move more slowly than the shorter fragments. So you end up with bands of DNA in the gel that you can see. Each organism will have the restriction sites at different locations so they produce different banding patterns. You can use this to determine how closely one organism is related to another.

So what it would tell us is how closely strain A is to strain B, and so on. I guess we could also build a restriction site map from the combined data.


-FF

P.S. Happy to clear that up for you, I'm not trying to be a dick, but it would've been easier if you just googled "gel electrophoresis".


--------------------
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

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Offlineebass
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584600 - 05/03/06 06:35 AM (17 years, 10 months ago)

OK thanks. So by using a restriction endonuclease on the DNA of different species one could find slight differences in base pair size.
How much of a difference in kb can one detect with a do-it-yourself kit? Also can changing the charge across the blot enhance any differences in RFLPs?
Sorry for the irrelevant questions...it's been a while since I've taken bio.

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5584706 - 05/03/06 07:44 AM (17 years, 10 months ago)

Most agarose gels are made between 0.7% and 2%. A 0.7% gel will show good resolution with large DNA fragments (5–10kb) and a 2% gel will show good resolution for small fragments (0.2–1kb).

I'm assuming that he will use around 1-1.5% gel and a 5 or 10kb ladder and so get around 5kb resolution or better. Maybe 2.5kb? I would be satisfied with that. RFLP tends to produce fairly large changes in fragment length. It will be really interesting to see the differences in strains. I would bet that only about 25% of the strains will have unique patterns.

Changing the charge orientation across the gel is only done for really fancy setups where you need to separate very large pieces of DNA. I don't really know that much about it.


-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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584721 - 05/03/06 07:54 AM (17 years, 10 months ago)

What strains do you think will be the most unique? Probable the SE asian strains vs GT or Acadian coast will show most.

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Invisiblefastfred
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5586507 - 05/03/06 04:18 PM (17 years, 10 months ago)

I'd assume that the more geographically distant strains would show the greatest variation from each other. That's just based on the assumption that geographical distance would be somewhat correlated with phylogenetic distance and that the RFLP would reflect that. But who knows, maybe some areas are more prone to mutation and genetic drift and would promote clusters of variability.


-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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5586798 - 05/03/06 05:27 PM (17 years, 10 months ago)

isn't genetic drift due to chance events?

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: ebass]
    #5588484 - 05/03/06 11:51 PM (17 years, 10 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.

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InvisibleBlimeyGrimey
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5589315 - 05/04/06 08:43 AM (17 years, 10 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.

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Invisibletexas34
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
    #5589498 - 05/04/06 10:20 AM (17 years, 10 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.

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
    #5593123 - 05/05/06 07:47 AM (17 years, 10 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5593753 - 05/05/06 11:43 AM (17 years, 10 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)

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
    #5599935 - 05/07/06 02:04 AM (17 years, 10 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."

:werd:

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5604546 - 05/08/06 11:03 AM (17 years, 10 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: texas34]
    #5631263 - 05/15/06 02:55 AM (17 years, 10 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5915383 - 07/30/06 03:56 PM (17 years, 7 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??? :cheers:

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.


--------------------
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
    #5916813 - 07/30/06 10:24 PM (17 years, 7 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.


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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: cloudtop]
    #5917715 - 07/31/06 04:22 AM (17 years, 7 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5917791 - 07/31/06 06:04 AM (17 years, 7 months ago)

Remember, its probably not in many vendors intrests to confirm that all the strains of P. cubensis are genetically identical. :grin:

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! :thumbup:

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5917939 - 07/31/06 08:19 AM (17 years, 7 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5918043 - 07/31/06 09:22 AM (17 years, 7 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 :smile:

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: TygerClaw]
    #5920985 - 08/01/06 03:58 AM (17 years, 7 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

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #5937597 - 08/06/06 04:32 PM (17 years, 7 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. :grin:

....




I was hoping it would appeal to a truly dedicated vendor, not some schlocky spore-jockey.  :wink:

There are some vendors that seem to have their hearts in the hobby.


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
    #5938250 - 08/06/06 08:29 PM (17 years, 7 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.

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: SpookerShroom]
    #5959443 - 08/13/06 02:43 PM (17 years, 7 months ago)

So we need someone to do the work, and someone to put up the funding.

Anyone think that FMRC might be interested???


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
    #5965718 - 08/15/06 10:58 AM (17 years, 7 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...


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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Herbus]
    #6040025 - 09/07/06 09:38 PM (17 years, 6 months ago)

k here's an update on this project.
firstly, my spore collection has been trashed, long story but putting your prints in a small lockbox is a bad idea, especially when someone steals that box (probably thought it had money in it, and i think i know who took it).

anyways, i now have the funds to order the EF kit to start this work. I have malabar spores to start with, but i believe this wouldnt be a good baseline to start from. i plan on using EQ or Gulf Coast as a starting point for comparison. i will be comparing the first sample to the PF Redspore (2nd sample) because i believe there is a good chance of noticing a difference between the two.
i'll post some more info one the kit gets here and i begin the work.
i do however need spores and some help researching the best enzyme(s) to use for spores. as stated before i want this research to be published and i dont want any legal trouble so live samples are out of the question.

i have however decided to also do a few different species (p. stuntzii, p. cyan, and p. azure come to mind) just for fun.

i will foot the financial end of this project (the EF work), but once again i'd greatly appreciate anyone who would like to donate spores or help with research.

anyways,
lets get this ball on the roll.

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: BlimeyGrimey]
    #6047578 - 09/10/06 06:13 PM (17 years, 6 months ago)

I'm also wondering about the variation to be found within one 'strain'... potency/yield etc.... I think we really need more examination of this area.


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: No_Life_G33k]
    #6048959 - 09/11/06 02:47 AM (17 years, 6 months ago)

Awesome to see this thing get going. As far as enzymes... I would just do a few searches on what has already been used and pick one of those. There has already been a ton of RFLP work done on various fungi.

It should be a pretty easy task to choose some good candidates. If you need any help feel free to PM me. You really can't go wrong here. EcoR1 and BamH1 are the most common restriction enzymes that would be on hand, so I would be tempted to use those and would be surprised if they weren't the ones in common use for RPLP.


-FF

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #6049995 - 09/11/06 01:34 PM (17 years, 6 months ago)

this is an interesting idea... sounds like alot of people have access to labs... excellent, i myself work in a phytochemistry lab... i don't remember who it was who said it, because i only have a limited amount of time to do this, i'm a busy man these days, which is why i am probably a stranger to all of you, but anyways, i think that the variation will be much much much less than 25%, most genetic variance between related species is very minute let alone mr's of the same species but different races? anyways i think we have the beginnings of an interesting line of inquiry here

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: PaulyPoor]
    #6055211 - 09/12/06 09:09 PM (17 years, 6 months ago)

Hey BliemyGrimey - I sorted out things with my lecturer and I can also do an RFLP; so we can combine our results.

I will carry out RFLP's between the members of the Psilocybe genus - and you already look set to do comparisons between cubensis strains so we have both bases covered!
I already have a reasonable (but small) selection of different Psilocybe species, so I'll get right onto sourcing some of the others... :laugh:

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: Feelers]
    #13548152 - 11/27/10 01:08 PM (13 years, 4 months ago)

did anything ever come of this?

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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: IGNVS]
    #13551758 - 11/28/10 08:54 AM (13 years, 3 months ago)

Wow it's been a while since I posted in this thread. In the end Sporeworks came through (very generously) and sponsored me with many spore prints to try out. I tried to actually get the Large ribosomal subunits sequenced, but I was told that it was too expensive by my lecturer.

To do all the species I had was going to cost around $600, the budget for the entire class was $1200. I asked him how much of that money had really been spent and he said less than $200, but that it wasn't fair to the other students. I eventually offered to pay for it myself, but he said I didn't know how to use the sequencer/pcr equipment. When I found a PhD student who was going to help me, the lecturer was still dead set against it.

Funny because I now have my own PCR!

All was not lost however, I revamped my experiment to utilize other expensive things that could be done in the time remaining, and jumped on the electron microscope.
These threads...
Here...
And here...


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