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OfflineFeelers
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Total Unrestricted Access to University Lab - What experiment??
    #6029820 - 09/04/06 09:39 PM (17 years, 8 months ago)

I realise I have posted vaguely about this before, but I didnt realise the scope that I was allowed to use things, and the project has just started.

I have full access to the electron microscope/lab, although I have to ask for it (I'm only 3rd year), and I can also order in chemicals etc.
I can do a project on whatever I wish, and since these opportunities are probably rare for us I want to ask for input.

I have a schedual of 7 weeks (not very long I know) and spores I have currently ... Ps cubensis, Stropharia Rugoso-Anulatta, Pan goliath, Cambo and Florida, Ps subaeruginosa, Ps. atlantis, Ps aucklandii, an unknown Psilocybe from inski, and a morel print.

I was thinking working with the pans would be awesome, but I am desperately trying to work out a protoplast fusion between Stropharia and Cubensis, but please put ideas out there.

So out of any experiment (although I dont want to get into genetics) what would you guys suggest? :laugh:

Workman, RR?

Edited by RogerRabbit (09/05/06 07:40 AM)

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OfflineFeelers
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6030449 - 09/05/06 05:05 AM (17 years, 8 months ago)

Ok I've been sussing out the Protoplast fusion side of things - I need a viable experimental method to present to my lecturer.

To start with - I have spores. I will germinate both species on PDA agar (with antibiotic) and use a microscope and disection needle to remove individual monokaryon colonies and transfer them to their own PDA dish.

SO I will now have plates of Ps. Cubensis and Stropharia rugoso-annulata monokaryon myc, (and I will check for clamps again for confirmation). The first problem I encounter - how old should the monokaryons be? I've seen some articles say 10 days, others say 4. Do I transfer the myc to a liquid medium for filtration later on? What I really need is a step by step process. MYA agar looks like it would be good to transfer to...

Link on protoplast fusion technique

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OfflineWorkmanV
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6031356 - 09/05/06 01:24 PM (17 years, 8 months ago)

I don't think 7 weeks is enough time for a protoplast fusion attempt, especially if you have never done it before. Just finding the right combination of enzymes for each species is time consuming. And there is no guarantee that the fusion product will even amount to anything. They generally don't fruit well or at all.

I am trying to think of a more feasible experiment, but nothing is coming to me at the moment.


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Invisiblefastfred
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6031512 - 09/05/06 02:10 PM (17 years, 8 months ago)

7 weeks might be a bit of a crunch. Regenerating protoplasts sometimes takes a while.

I can recommend the book mentioned in the link you posted. It's quite informative and a good read. "Genetics and Breeding of Edible Mushrooms". It has a lot of coverage of protoplast fusion.

If you're looking for a more in-depth book with more numbers then check out "Fungal Protoplast: A Biotechnological Tool" by D Lalithakumari. It's not that good of a read, but it has more details and doesn't force you to go to the original papers as much.


> The first problem I encounter - how old should the monokaryons be?

They should be "young". See page 17 of Lalithakumari (Age of the culture). I've seen papers using everything from 10hrs to 14 days. As long as the myc is still in log phase I don't think it'll make or break you. Protoplast yield does drop off the older the culture gets though.

> Do I transfer the myc to a liquid medium for filtration later on?

Yes. The whole process is done in liquid until the protoplast regeneration.

> What I really need is a step by step process.

Look up a paper and that's what you'll get. The "Genetics and Breeding of Edible Mushrooms" book has lots of good citations.

> MYA agar looks like it would be good to transfer to...

Most papers use MCM (mushroom complete medium) for protoplast regeneration. I would use PDA or MEA rather than a chemically defined medium like MCM. I would avoid yeast extracts.


OK, so I realize that you're not wanting to do genetics, but doing an RFLP would be easy and produce some good results. Since you have spores from a few strains it should produce some good and interesting data. All you'll need is a DNA extraction kit and a standard restriction enzyme or two. It's pretty cheap and simple, and you're guaranteed success.

Another interesting thing to do would be to spec different spore solutions. That would be really simple and easy and require nothing. It would be interesting to check the differences between the redspored cubes and the normal colored ones. I've done a couple, but haven't had anything really interesting to run.

All in all I think your protoplast fusion ideas aren't well developed enough and/or are too ambitious. You didn't mention any methods for selection of fusants, and that's really the hard part.

I'll let you in on a good idea I had. This should cut out most of the hard steps. There are some strains of fungi that have mitochondrial resistance to certain fungicides. I believe that you can even get auxotrophic strains of these strains. So just find something with mitochondrial resistance to some fungicide which is also auxotrophic. Then you will be able to easily select for fusants. You'll be able to easily "brute force" your experiment since you'll have a good way to select fusants. The mitochondrial nature of the trait will also make it much easier and more likely to succeed.

Otherwise you're stuck with nutritional complementation, which means that you'll need to develop auxotrophic strains of both parent strains. That isn't TOO hard, but it's not doable within your timeframe. With my way you can just grow on minimal media with fungicide and it will eliminate all non-fusants.

Of course you won't end up with a super-hybrid, but you would get a mushroom with fungicide resistance, which would be pretty handy IMO. Check ATTG for the strain you need.


-FF

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: fastfred]
    #6031656 - 09/05/06 02:38 PM (17 years, 8 months ago)

I think I may only have six weeks - and its supposed to be done in 5, he was confused about when our lectures/exams finished. :frown:
So thats even worse than I thought. One thing though - I could just do the fusion and not worry about fruit bodys - ie just get transformed myc.

But it does seem off the cards now, so onto other ideas. I am keen to use the Pans somehow - I have 3 different strains on hand??? Oh I also have C. comatus also just remembered.

How about a Subaeruginosa vrs Aucklandii interaction? I could do an SEM of the "battle". I really need to get my shit together :laugh:

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6036478 - 09/06/06 08:41 PM (17 years, 8 months ago)

RR you edited my first post? Not worried just curious?

Anyways... OK FastFred, I might be convinced to do some genetic relatedness-type things, I havent asked about this aspect but as I do have quite a variety of species so it could be good.
Can you post some more info on this please? :laugh:

I will start cultures of everything I have so far tomorrow, and if some more ideas pop up the cultures will already be fired up and ready.

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Invisiblemonstermitch
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6038054 - 09/07/06 10:57 AM (17 years, 8 months ago)

Quote:

Feelers said:
Workman, RR, FastFred?




to

Quote:

Feelers said:
Workman, RR?




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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: monstermitch]
    #6048986 - 09/11/06 03:40 AM (17 years, 8 months ago)

Wow! It's sure nice to see that RR has become a mod. It's also good to see that he's not abusing his power or anything.

The short and sweet of it is that RR hates me. I've pointed out a few issues that he's been dead wrong on and he's extremely bitter about it. I've tried to be nice about it, but some things I just can't let slip. I couldn't help but correct his claim that shiitake produce non-viable spores and now he's paying me back for it.

I don't think I like where the shroomery is heading. I recently got banned from cultivation for 14 days just for mentioning the fact that a robotic harvesting thread got closed in advanced. It was in response to someone in cultivation who asked about how people harvest mushrooms. I mentioned that some people have used robotic harvesters to individually pick mushrooms using AI, cameras, and robotic arms, but that the thread on it in advanced got closed because it was too advanced or not anvanced enough, or something. It was claimed that I was "baiting" someone and that was used to justify a 14 day ban.

So just a word of warning monstermitch... Apparently it is now against the unwritten rules to even mention anything that has gotten closed or changed or rewritten without the owners consent.

And of course mentioning what the unwritten rules are is surely a cause for banning, so good luck to everyone, I'll miss you when I likely get banned for this post.


> Can you post some more info on this please?

I'd be happy to help. I have a major time squeeze at the moment though. There is a ton of published literature on the subject, I'm sure you can find several good protocols. I've found that the biotech companies are also happy to help you find use for their products. If you were to start with a DNA extraction kit from Quiagen or the like, I'm sure they would be happy to help you carry it through to good gel results. If you run into any problems they are usually very happy to help you resolve them and trouble shoot your process. They also would certainly have a recommended protocol for their kits. That's probably what you're looking for... a step by step procedure to follow.

The procedure is not complicated at all. You simply purify the DNA (usually and most easily done with a standard purification "kit"), load it into a gel with some loading dye, load a standard DNA ladder on either side, and apply electricity across the gel with your power supply. Then you scan in the gel or take a picture of it (on a transilluminator if your using ethidium bromide) and you're pretty much done. Then you just have to do some comparisons and make sense of the banding pattern. It's pretty basic and is a good project for an undergrad.

There are different ways to go about it. You have a few parameters to figure out. You need to know how pure your DNA is in order to know how much to load in each well. You also need to know approximate sizes so you know how much voltage to apply and for how long. Some smarty-pants types might try to tell you to do a lot of fancy calculations, but the standard method is to just load a batch and go with it. Just don't run your loading dye off the end of the gel. If your bands are too blurry then you've used too much DNA or run at too high of a voltage. If the bands are too faint then you didn't use enough DNA or dye. But don't worry! Just use standard numbers and 90% of the time it will turn out fine. You can then fine tune it or just keep doing it the same. If it doesn't seem to work at all then ask for help, there aren't that many things that can go wrong. One bit of advice though is to spec. your DNA to find the concentration. Just find a decent spec and it will usually have a DNA concentration function that will make life easy for you. But if you follow the kit directions you'll get a fairly expected DNA concentration so don't worry. PM me or post if you have any specific questions.

Since you're in a bit of a time crunch I would suggest doing the RFLP. Most of the supplies should be on hand, everyone will know what you're talking about and can help you, and you can do the whole thing in one day. It would be a major advance for shroom science. You might try to see if the vendors would help you out with spore samples, it would be in their interest.

Oh, I almost forgot... this has been discussed before. Here is the thread...
http://www.shroomery.org/forums/showflat.php?Cat=0&Number=6048959&page=0&vc=&PHPSESSID=#Post6048959


-FF

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OfflineFeelers
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: fastfred]
    #6049021 - 09/11/06 04:46 AM (17 years, 8 months ago)

Don't worry FF - I still love ya! :smirk:
RR I would like an explanation on the edit - I have a lot of respect for both of you and I really dont see why "FastFred" should have been removed from my post.

But back to business...
Yeah I saw that other thread - we can probably add our results together.  So - I what am I gonna test - all the species I have?? I was thinking P. cubensis strains would probably be the best - but I only have access to two strains - Huatuala/guadajalra (or however you spell them, cant remember which one) and B+. They have a fairly good genetics lab I think here so they should have all this stuff easily available.

I will talk to my lecturer tomorrow about what can be done. :laugh:

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: fastfred]
    #6049022 - 09/11/06 04:47 AM (17 years, 8 months ago)

Im not sure of the background with that but i think its obviously no good to ban someone simply for mentioning that they got another post deleted.
God a year ago i was regularly getting in arm wrestles with people - including mods and never had any trouble at all.

As for access - my interests are probably more in the lines of isozyme protein analysis and things along those lines to map specification, etc.


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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Zen Peddler]
    #6049023 - 09/11/06 04:49 AM (17 years, 8 months ago)

Can you explain some more BM? I am keen to do something advanced, if I have all this stuff available for a short time I really wanna make the most of it.

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6049035 - 09/11/06 05:09 AM (17 years, 8 months ago)

well its kinda like genetics i guess - simply checking the spore compatibilities of different collections and comparing isozyme proteins basically to evaluate whether collections are cross compatible or different species


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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Zen Peddler]
    #6049920 - 09/11/06 01:12 PM (17 years, 8 months ago)



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Invisiblefastfred
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Zen Peddler]
    #6049991 - 09/11/06 01:33 PM (17 years, 8 months ago)

> God a year ago i was regularly getting in arm wrestles with people - including mods and never had any trouble at all.

Yep, me too. But the times, they are a changin'. Most of the mods are really cool here, but there are a couple that take things way to seriously and will ruthlessly snipe at you for past issues.

Some people just really hate being proven wrong. Rather than just admit they were wrong or simply shut up on the issue they get personal and resort to name calling, unfair censorship, or other childish antics.

What really gets me is that if you point that out or even mention any sort of displeasure with that sort of fascist BS, then they accuse you of "baiting" whatever that means. It seems like it's a catchall excuse they've come up with for banning any sort of dissident.

I better not say anymore on that, lest I get banned. I'm sure they're already discussing whether to ban me in the mod forum.

> Can you explain some more BM?

"Genetics and Breeding of Edible Mushrooms" has some good sections on isozyme analysis. It's another technique along the lines of RFLP. Basically the idea is that the same enzymes in related species are slightly different and you can make comparisons based on those differences.

DNA -> RNA -> Protein, so small changes in the DNA result in small changes in the structure of the protein produced (the enzyme). If the change doesn't change the function of the enzyme greatly and it's still functional then it gets passed on. In that way enzymes develop different structures.

To do isozyme analysis you would usually run an extract through a column to separate out the different sized proteins, then you would run a certain fraction on a protein gel which would give you a banding pattern somewhat like an RFLP. It's handy for verifying protoplast fusion or mating, but not quite as handy for genetic comparisons. Protein electrophoresis is also more expensive and the whole process is more complicated.

> Yeah I saw that other thread - we can probably add our results together.

That's the beauty of it. Any results you get will serve as the basis for all future comparisons. Nailing down and optimizing a good procedure will also pave the way for others to do the work without worrying about developing the protocol. Of couse, it should be pretty easy and pretty standard, but it will still be helpfull for others to follow without having to do any research.

I would try to do as many species and strains as possible. You might as well do everything you have on hand. As soon as you are sure that your going to do RFLP I would ask around for as many samples of various strains as you can get. You can easily do 10 lanes on a standard sized gel. If you load ladders in either side that's 8 lanes left for samples. You'll probably do more than just one gel, so you should be able to do a fair number of samples.


-FF

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: fastfred]
    #6051635 - 09/11/06 09:27 PM (17 years, 8 months ago)

Sweet! Good news, I had a talk with my lecturers and the technicians and I can do an RFLP - and I can use the automated Gel-Doc system, like this thing... Gel Doc System
to do the counting for me, and in high resolution. :grin: :grin: :grin:


She also said I might be able to use the DNA sequencer - would I be able to sequence the genes responsible for psilocybin production using this? I dont really know shit all about all the different techniques, but I'm learning. She also mentioned an ALP aswell, didnt catch much of that though.

I'll need to get my sources of spores sorted, it looks like I will be able to do a few Gymnopilus species also.

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OfflineRogerRabbitM
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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6051686 - 09/11/06 09:39 PM (17 years, 8 months ago)

You would need a DEA permit to do that if you're in the US I believe.

I agree with workman that seven weeks is far too short a time for protoplast experiments. You could do an excellent paper on genetics by mating monokaryons from various strains of edibles. I'm jealous of the pictures you'll be able to get.
RR


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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: RogerRabbit]
    #6051754 - 09/11/06 09:56 PM (17 years, 8 months ago)

Nope not in the US - all those fundamentalists would drive me nuts! :laugh:

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Invisiblepoke smot!
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Re: Total Unrestricted Access to University Lab - What experiment?? *DELETED* [Re: Feelers]
    #6052586 - 09/12/06 08:03 AM (17 years, 8 months ago)

Post deleted by poke smot!

Reason for deletion: x


Edited by poke smot! (09/12/06 08:09 AM)

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: poke smot!]
    #6055254 - 09/12/06 09:20 PM (17 years, 8 months ago)

OK after seeing BlimeyGrimeys experiment on the difference between cubensis strains, I will carry out anaylsis between the Psilocybe genus.
So I have so far... Ps. cubensis, Ps. subaeruginosa, Ps. Atlantis, Ps. aucklandii, (three strains of Pans that I'll include) unknown Ps. from New Zealand.

So I need/want... Ps weillii, Ps. mexicana A/B, Ps. azurescens, Ps. cyanescens, Ps. Hispanica, Ps. makarorae, Ps. semilanceata, Ps. caerulipes, Ps. Tampanensis.

Can anyone add to this list? I know there are quite a few missing, I might get a hold of cactu...

If you have a print that you would like to donate (the print can be very faint or even a fragment of a print(I have agar there is no problem)  PM me! :laugh:

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Re: Total Unrestricted Access to University Lab - What experiment?? [Re: Feelers]
    #6056024 - 09/13/06 01:58 AM (17 years, 8 months ago)

You might be interested in these threads...

http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5748260

http://www.shroomery.org/forums/showflat.php?Cat=0&Board=Forum4&Number=5707531

That's a nice gel doc system you have access to there. You'll probably end up producing the first AND the best RFLP gels.

If you want to do some sequencing then check out the above threads. The primers for the LSU region are published in the literature, so that would make doing the sequencing and phylogenetic analysis a snap. I don't think you'd find enough variation in the LSU to be very useful for intraspecies phylogeny, but it would be nice to add some of the species you have at your disposal to the tree.

Another way to get slightly fancier than RFLP would be to use RAPD (Random Amplification of Polymorphic DNA) or AFLP (Amplified Fragment Length Polymorphism). Both are fairly common and reasonable to do. Good luck and be sure to keep us updated.


-FF

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