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Re: IMPORTANT, I need a few answers!!! [Re: Feelers]
    #6145911 -

Ok I need some help with the PCR conditions.

I will be using this method to extract the DNA...
Quote:
Vegetative spores from 13 Psilocybe species were taken from spore prints with a loop and transferred to empty 500 ul Eppendorf tubes. Each tube was closed and the dry spores were irradiated in full power (700W) for 5 min in a domestic microwave oven . A beaker containing water was present during the irradiation to avoid damage to the oven. Thirty microliters of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) was added to the tubes, the tubes were then vortexed and spun in a microfuge for 1-5 min at 14,000 RPM. The supernatant liquid was collected and transferred to a clean Eppendorf tube.


SO I take 5ul of the resultant DNA extraction, and I need to PCR it with the primers. I've never done anything like this before so I'm not really sure what to do...

What additives do I add? How much of the primers do I add for this PCR ? I think I worked out that TAQ is dna polymerase, so I obviously need to add that - how much etc.

The conditions are quoting from that original phylogenetic alalysis of psilocybe...
Quote:
The temperature program is given as follows: 94 °C, 4 min; 30 cycles of 98 °C, 15 s; 55.2 °C (for ITS) or 62 °C (for LSU), 5 s; 74 °C, 30 s; 74 °C, 4 min.


But they didnt go into detail about how much TAQ or primers they used, would the process change dependent on what additives you put in?

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Re: IMPORTANT, I need a few answers!!! [Re: Feelers]
    #6147158 -

> Ok I need some help with the PCR conditions.

There are lots of variables. I don't want to give specific instructions that might not work for you. 98% of doing science is finding the closest paper to what you're doing and replicating it exactly. So I advise that you study the literature well.

I don't know anything about the microwave extraction procedure. It seems to me that there are a lot of unknown variables in it. Every microwave is different and there are lots of hotspots and coolspots inside that get different amounts of energy delivered. The amount of water in the beaker would also affect how much energy is delivered. I've always just used extraction kits or followed standardized procedures, so I don't know much about other methods.

I would suggest following the microwave paper if that's what extraction method you're going to use. If you follow the paper the first time you can then make slight adjustments to the procedure based on your results.

Here is a good site on PCR troubleshooting and tweaking.
http://info.med.yale.edu/genetics/ward/tavi/Guide.html
http://www.med.yale.edu/genetics/ward/tavi/Trblesht.html

It gives answers for your questions about TAQ and primers, etc. Your main variable will be how much DNA you actually have in your starting mixture. So after your extraction procedure you should use a spectrophotometer to determine your DNA concentration and then dilute your sample or use less of the primers and TAQ as required. Otherwise you could just go with whatever the paper did, but your results will vary a bit then if you don't account for the amount of DNA.


-FF

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Bugger [Re: fastfred]
    #6148421 -

Arrrghh!!!!

They decided they arent going to buy me the primers, so from that standpoint I'm fucked. I offered to buy the primers and pay for the sequencing also and they said no!  :crankey:

So that means that all my write up so far has to be abandoned, which I'm real fucked off about. It sucks because the lecturers (as good as they are) dont know much about the PCR side of things. There is a group of postgrads that have been helping me out, who deal with PCR, and while they think there will be problems, it should work in the end, my design is better than most because I already know the primers and PCR temps required. They even offered to guide me through the procedures and still I get the big NO.

However supposedly I can apply for a summer scholarship to have this carried out. Due to the legality of having all these spores I'm not really sure if that would be approved. At the very least I would be able to sequence aucklandii and subaeruginosa, but I spose I could just lie about the others.
SO just in general I'm fucked off. I really don't understand why this happened, talking to some of the post grads they said that a few years ago people in this class with whacked out projects were encouraged, but now they give you a fungus - and you are expected to do the normal growth rate per day type cabbage science.

So I'll see if I can get the scholarship thing - it would be pretty rad, $3000. I'll see how that goes, in the mean time I have to do a real crap filler in project but perhaps you guys can help me with that, it'll probably be using my electron microscopy pics. I might ask Sporeworks for some funding to do the sequencing, I'll still try to get it done but I cant use it for my project. I'll be happy to return all the spores to people that I've recieved. I'll keep this going, at the very least I will have some more SEM pics to keep everyone entertained.

What a throughly depressing day! :frown:

Add to that I found out that some nice cologne I ordered appears to have been lost in the mail - for a second time. The first one was lost between Hong Kong and here, and the second one made it to the Depot, but noone knows where it is! I think I'm gonna go home, crank up some Meshuggah (the mouth licking what you've bled) and smash some of the old furniture we have round back.

:grin:

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Re: Bugger [Re: Feelers]
    #6149205 -

How about just doing compatibility and isozyme protein studies??


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Re: Bugger [Re: Zen Peddler]
    #6151815 -

I did look into an RFlP but you need to PCR for that also I believe.

Since I'm now seriously worried about handing in a decent project (this is worth 45% of my year) within the next two weeks I'm gonna put the genetic thing on the back burner, my new "cabbage project" will be a electron microscopic comparision between fruiting bodies of Amanita muscaria and Psilocybe subaeruginosa.

OH some good news at last! - The three way battle between Ps. cubensis , Pan cyan (goliath) and Ps. atlantis worked, so will get some pics of that for you guys.

AND!!! would you believe some absolute monkey in my lab changed the temperature on the 25C(in huge writing) incubator up to 40C to do their experiment despite the fact that everyone else was using it also. So thats why all my isolates have been acting wierd, my subaeruginosa isolate is incredibly dense and my aucklandii isolates are tiny but an odd shade of brown (but looking healthy none the less).
Its funny; that person must have screwed up at least ten peoples projects, I think the temp has been set like that for at least a week, possibly even the whole time. Interestingly atlantis is by far the best grower, it seems to like it hot! The pan cyans look very weird, almost forming little dense balls of myc within the very very wispy fluff.
What a fucken spoon, no wonder they don't think I can do a PCR. :grin:
Anyways, hold your breath, I have a feeling my next pics are gonna be awesome! :thumbup:

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Re: Bugger [Re: Feelers]
    #6152805 -

RFLP doesn't require a PCR. It's one of the simplest things you can do. It's as easy as any cabbage project, so I can't see anything they'd object to.

You just extract the DNA, cut it with restriction enzyme(s), load into a gel and run. It's really that simple. Restriction enzymes, unlike primers, are completely standard and your lab will certainly have some on hand. You might try to do a couple since there is really no way to fail and the entire procedure can be done in one day's work. If they won't let you do that it isn't much of a class. In genetics 101 I sequenced the d-loop mtDNA region for 2 different mouse samples. That's on par with sequencing ITS-1, 5.8s, and ITS-2.

If you try to sell them on the simplicity and quickness of an RFLP I bet they'd go for it.

That's wacky that somebody changed incubator temps! Especially since there's probably a 40C incubator already around that he could have used. Strange that nobody noticed that the 25C was so hot. Good luck with your experiments and avoid that dude.


-FF

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Re: Bugger [Re: fastfred]
    #6152855 -

I think everyone else was like me - I'd never have thought to check the incubator temp, its the technicians who sort all that stuff out. There was a 40C incubator else where which was why it was so dumb!

Hmmmm, how do you interpret RFLP data from the babding patterns? If I use the gel doc system can that work it out for me?

I will say I'm still intrigued.

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Re: Bugger [Re: Feelers]
    #6152888 -

Wait remember that I only have spores - I'm unsure of whether I could extract enought DNA for it to show up, and I dont know whether that microwave extraction would be ok for that?

I know restriction enzymes are common place but when I asked if they had any generic restriction enzymes she said - no they are all site specific. Well duh I mean ones that are used by everyone!

I'll ask again tomorrow, I got the feeling that they wernt keen on the genetic side of things.

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Re: Bugger [Re: Feelers]
    #6153123 -

You do have some specimens as well dont you?
I would go with the RFLP.

And aucklandii mycelia is a yellowish colour that browns with damage.


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Re: Bugger [Re: Feelers]
    #6153209 -

Feelers said:
I know restriction enzymes are common place but when I asked if they had any generic restriction enzymes she said - no they are all site specific.


Ha ha... That's a pretty good joke! I assume she was joking or being a smartass. Of course they are all site specific! If they just chewed up your DNA into bases they wouldn't be much use would they.

Just tell them you need 50ul each of EcoR1, BamH1, and HindIII. Those are the three most common ones and they are sure to have them on hand.

I don't know much about the microwave method, but you don't need all that much DNA to do a RFLP. You only have to load one lane for each sample, so I can't imagine it would take that much.

There are lots of ways to interpret RFLPs. The simplest is to just look at the banding pattern and say same, similar, or not similar for each lane or gel you run. To go further you can look at each band and say weather it's present or not in each sample. You can also compare the size of fragments. So on and so forth. If you get real fancy you compare different restriction enzymes on the same sample and also do partial digests and you can create a map of the restriction sites.

But to start you would just say sample 3 and 5 have 5 bands in common, two bands different, and one that's changed in size. As an example.

Not too difficult.


-FF

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Re: Bugger [Re: fastfred]
    #6154007 -

Nah man she was serious! And the thing is it looked like I didnt know what I was talking about because she then explained how one worked. :grin:

How accurate have phylogenys been; based on these RFLPs?

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Re: Bugger [Re: Feelers]
    #6156441 -

Well I got some more pics on the SEM, the battle came out real awesome!

:grin:

To be posted soon...

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Re: Bugger [Re: Feelers]
    #6156862 -

Sorry BM I tried to get a look at your unknown dung-lover species but the guy said it was a bit far gone to see anything.

He was in a rush so I dunno, it was quite pretty chopped up unfortunately.

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Re: Bugger [Re: Feelers]
    #6156868 -

> How accurate have phylogenys been; based on these RFLPs?

Pretty accurate. It operates on the same principle, just uses a different way to visualize it. When you sequence something you compare the sequence and see how similar it is. Restriction enzymes cut at specific sites, so any change to those sites will prevent a cut at that location.

So if you know there are 4 bands in the size range you are looking at then you know that the restriction site is (most likely) repeated 8 times. If you compare that to less bands you know that some restriction sites have been eliminated or inactivated. So using that you can make a comparison. You can also look at band size changes and see how much (or how many times) there have been insertions or deletions in that chunk of DNA.

So the theory is the same (comparing changes in DNA), but the analysis is less straightforward. The analysis is different, but is no less valid as a way to compare species.

Some would argue that sequencing is better because it gives concrete data that is easily analyzed by a computer. But I would point out that there are advantages to RFLP. Sequencing compares a small section, while an RFLP examines sites spread across the genomic DNA. It can also show up large insertions and deletions, whereas sequence comparison usually completely discounts those during the alignment step. I'm not completely sure on that, some programs may give some value to those insertions or deletions, but the ones I've used usually just align the sequences and produce a sort of alignment of the sample vs consensus sequence then compares that sequence to the others.


-FF

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Re: Bugger [Re: Feelers]
    #6157228 -

LOL yeah the condition of that one was disappointing. maybe next year. After considerable study under the scope I should have sent you the SA subaeruginosa varieties. Their cystidia are more different than Ps.cyanescens and Ps.subaeruginosa from Victoria - quite unique. Anyway have fun! :wink:


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Re: Bugger [Re: Zen Peddler]
    #6157300 -

Has anyone ever figured out what cystidia and plurocystidia actually do? I can see how plurocystidia could help hold the gills from touching, but is that all?
RR


--------------------
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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Bugger [Re: RogerRabbit]
    #6158378 -

Here's the thread I'm gonna post my electron microscope pics in,
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/6156471/an/0/page/0/gonew/1#UNREAD
First on is a microscopic Atlantis sclerotia that's just formed. RR there are some visable clamp connections in some of the pics. In the "battle scene" you can see one of the species hyphae wrapping around the attacker.

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Re: Bugger [Re: RogerRabbit]
    #6158471 -

"cheilocystidia serve an excretory function, so the margin of gills with abundant cheilocystidia are often beaded with tiny droplets of liquid."

Cystidia might serve the same function. It might help regulate moisture content of the gills or gill surface. Maybe to keep gills moist until the spores are developed, or maybe to help dry them out so that the spores drop at the right time.

That would be my guess.


-FF

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Re: Bugger [Re: fastfred]
    #6158604 -

Do you have the reference for that FF?

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Re: Bugger [Re: Feelers]
    #6159948 -

I updated my pictures thread - you guys might be interested in the "battle" it came out pretty cool.

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Mushrooms, Mycology and Psychedelics > Advanced Mycology
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