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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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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, 8 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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Offlineebass
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Re: The Shroomery Strain DNA-Length Analysis Project! [Re: fastfred]
    #5584721 - 05/03/06 07:54 AM (17 years, 8 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, 8 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


Extras: Filter Print Post Top
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