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PCR and Electrophoresis for ID and Cordyceps mating type ID * 2
    #26511926 -

First of all i would like to mention and thank William Padilla Brown and Alan Rockefeller for all their assistance and knowledge. Will wrote an excellent Article in Fungi Mag on this i strongly recommend you check out. 

Cordyceps Militaris require two opposite MAT types in order to breed and produce fruit bodies. (Bipolar heterothallic ascomycete) there are 3 different "sexs" within C.M,
you have your "MAT 1-2-1" aka MAT2
then you have MAT 1-1-1 & MAT 1-1-2 both of which are called "MAT 1"
so while there are 3 "genders" really you just looking for mono cultures that are differnt from each other within those 2 MATS (1&2)
so for example MAT 1-2-1 can breed with 111 & 112 but not itself. mat1-1-1 cannot breed with mat 1-1-2 or itself.
ok i hope that makes sense. you need a "MAT1" and a "MAT2"

so without running PCR half of you breed attempts will fail, it can still be done just more time and materials, PCR is also useful for identification of Mushrooms and the steps are very similar.

for this example (mating Types) i ran 8 different monokaryotic cultures. you need 2 PCR tubes per culture so its a total of 16 samples.
I start by taking 8 centrifuge tubes and placing a small wedge of culture into each keeping track of which is which.
i then pipette 100ul of ES solution into each one, (be sure to switch pipette tips as to not cross contam) i then grind with a pestal (Sterilized) and wrap each one in electrical tape then drop into a ziplock bag with a weight in it then drop that into my digital thermostatic water bath @ 95c for ten min. its important to tape the tubes if they are the snap lids or then have a tendency to pop open in the bath. if you have the screw lid tubes you wont need to do this. after 10 min i pull them out remove the tape and add 100ul of DS solution (BSA and sterile distilled water at 3%) to each of the 8 centrifuge tubes and spin in the centrifuge until well mixed.
next we need to make our MAT 1 & MAT 2 master mix
so i need a total of 16 PCR tubes so i take 2 clean centrifuge tubes and label one of them MAT1 the other MAT2. to the MAT1 tube i add my 76ul of pure H2O
100ul 2x taq master mix
8ul of of my MAT 1
Forward primer:( MAT1-1-1-F:5'-ATGGAACACAGATCGAGCGACAC-3)
8ul of my MAT1
reverse primer (MAT1-1-1-R,5'-ATATACCTTCGCGATCA TTGCCCAG-3)

then to the other centrifuge tube labeled MAT2 add the same water and MM as the other but to this one add 8ul of each MAT2 primer
Forward: (MAT1-2-1-F,5'-TGTTTGTCGCGATGGTTCTGG-3')
MAT2 reverse(MAT1-2-1-R,5'-CCTCTGGAGGTTCTGCATTCCA-3'

make 2 rows of PCR tubes (8 tubes in a strip) one row will be MAT1 the other MAT2 add 20ul of your MAT1 MM to each of your tubes in the MAT1 row then do the same with MAT2 MM to the MAT2 row. then add 1ul from each of you 8 centrifuge into you PCR tubes. so the culture tube with your first mono culture in it that we prepped earlier will deposit 1 ul into your MAT1 PCR tube and 1ul into your MAT2 pcr tube be sure to switch tips inbetween and since 1ul is such a small amount its a good idea to put your tip into the MM in the PCR tube and pipette up and down a few times to be sure you get it in there. load all yout PCR tubes with your samples then close the lid tightly and its time to run PCR.
PCR protocol
95c 1min then 30 cycles of 95c for 30 sec 58c for 30 sec and 72c for 40sec then a finishing extension of 72c for 5 min

For ID the steps are very similar except you dont need to do 2 PCR tubes for each sample. ill do the math for 8 again since the strips come in rows of 8. now for ID you want to be sure you have enough left in the PCR tube after electrophoresis that you can send off to get sequenced. so ill do the math for 10 tubes.
10 tubes X 25ul reaction volume
250ul total
2x Master Mix
250 divided by 2
125ul mm
250-125ul(MM)-10 (F primer) -10 (reverse primer) -10 (sample)
95ul h2o
so
125ul of master mix
95ul h20

then add your water your MM 10ul of your Forward primer (ITS1F) and 10ul of your reverse (ITS4)
then put about 24ul of your MM+primers in each PCR tube containing the mushrooms you wish to identify
then you run PCR on these and the PCR protocol is a little different your gonna want to run
step 1 (denaturation) temp 95c for 3 min 
step2 (temp) 95c for 30 sec
step3 (annealing) 54c for 30 sec
step4 (extension) 72c for 59 sec
step 5 ( go to ) step 2 (35X)
step 6 (final extension) 72c for 7 min

after the PCR has finished its time to run electrophoresis!

so first you need to make your gel that is simple just take 75ml of 1x TAE buffer and h2o and mix with 1.5g of agrose, add in 8ul of gel safe DNA stain and microwave until clear. then pour into your mold with your slot ladders and wait to cool.

now we need to add your loading buffer dye to your PCR samples (unless your mm came pre dyed) to do this lay out dots of 3ul of loading dye onto para film then add 8ul of each sample. for cordyceps keep your MAT1 & MAT2 of each sample next to each other so its easy to keep track. drop your gel into your electrophoresis box cover in 1X tae buffer and pippet your sample (post dye) into the slots (1 sample per slot) be sure you are running towards the POS. i drop a DNA ladder in the last slot to give me a way to compare BP size. for ID the results are very easy to read. if you see your DNA band under the blue light it means that PCR worked and you can send the rest of the sample off to be sequenced. for mating types its a little more complicated. first off only one of each MAT (1or2) should show a band, if they both do it means that the cultures had already mated and it was not a mono culture. if only one band appears then you compare its size against the ladder. MAT1 is 457 BP long and should coincide with 5th band from bottom of ladder while MAT2 is 368 BP and will be closer to the 3rd and 4th ladder band. its really hard to get pic good enough to show the MAT type sizes but im gonna try to get a good one here soon.
here is a Mat1 and Mat 2 C.M culture

here is an ID run


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JOC PAN TEK
CORDYCEPS MILITARIS

EXOTICS
[

Edited by Jakeoncid419 (03/01/20 08:03 PM)

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: Jakeoncid419]
    #26512568 -

Thanks for posting this! Would you please tell me the source for the MAT primers you used? I'd also be interested to read anything on cordyceps genetics and breeding you can recommend.

A couple notes. You may not get the desired fidelity if you're running the PCR on raw mycelium. I've had good results extracting the DNA from 330 mg mycelium with NucleoSpin Soil kits from Macherey-Nagel GmbH.

What's the point of the water bath and the BSA solution? If it's to break the cells, please know that protein-bound metal ions in the cytoplasm will cleave the DNA and ruin it if they reach the nucleus.

Also, the PCR program will vary depending on your machine, the reaction volume, the thermal properties of the primers, the expected amplicon length, and the replication rate of the master mix enzymes (Taq should be ~1 kb/min). The NEB Tm Calculator is a great resource. You're also adding the MM at the last possible minute, right?

I stopped using Parafilm to mix gel stuff and now only use PCR tubes. It makes it so much easier to load the wells, plus you can close the tubes to prevent the dye from drying out. Also, the dye is properly diluted, right? Most I've seen are at 6× concentration, i.e., 1 µL dye to 5 µL PCR product.

Then you can cut the bands out of the gel and purify it using silica beads, ethanol precipitation, etc., to remove the leftover PCR shit like polymer dimers and other improperly annealed fragments.

Anyway, sorry to nitpick, I just want this to go well for you. A resource on cordyceps mating genetics that originated on the Shroomery would be a huge development for the community.

My notes are also for the benefit of others who may want to do their own similar work but don't have access to the same expertise, a lot of PhD biologists who know all about the gotchas and finicky bits for each step of the protocol.


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Edited by AndyHinton (03/02/20 10:24 AM)

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: AndyHinton]
    #26512909 -

Excellent I was hoping people would comment in and this process could be improved upon. As for the extract and amp kits that you speak of The ES & DS solution used is reversed engineered from those kits. Their ds is BSA & h2O & 3% and the ES is Extract-N-Amp Solutions Extraction Solution (ES) Stocks basically 1 M Tris solution (pH=8.0) 1) add 10 ml of 1 M Tris stock, KCl 3), EDTA 4) and DI or ultrapure H2O and shake until solutes dissolve, then titrate with 1 M NaOH to pH = ~ 9.5-10.0 6). All the extract and amps are specifically targeted towards plants however they do seem to work just fine but it’s more expensive to buy the kits.

I feel like using tubes instead of a strip will waste a lot of tubes.... I move pretty quickly on the parafilm I’ve never had issues with things drying out.
Why would I cut the bands out of the gel you mean to use the sample I ran through the gel as my sample I send off for Sanger sequencing? Are usually just make sure I have enough left in the PCR tube to send it out after I verify it worked (for ID)


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JOC PAN TEK
CORDYCEPS MILITARIS

EXOTICS
[

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: Jakeoncid419]
    #26512999 -

Interesting, thanks for the details. NaOH-based methods are fairly old tech, and I wouldn't recommend them (I'm also biased because my one DNA extraction attempt with NaOH was a miserable failure). It also seems tricky to correctly deionize the cytoplasm.

The Macherey-Nagel soil kit I mentioned uses silica beads and one of two lysing buffers designed for different soil humus contents. There's also multiple washing steps before you finally elute the bound DNA through spin filters. We ran the numbers and it was actually cheaper to buy the kits (I think it was either $150 or $300 for 50 preps).

They work really well, especially for pure mycelium without the contaminants inherent in soil or fruitbodies. I verified every extract with a Qubit fluometer and it was common to recover more than 25 μg/mL per sample, but the amount varied wildly. Either way, it's a really clean method, especially if you transfer the bead tube supernatant to a fresh tube when you bind the DNA.

I happen to have access to a well stocked lab so mixing the gel stuff in PCR tubes is no big deal. But you're right, it's less economical than Parafilm and swift movements. My words about cutting out the bands is for PCR purification. Most sequencing services can take unpurified PCR product for an extra cost.

But again, the BosLab is really well stocked because of its location. We have 2 or 3 gel purification kits with 150 preps each, that were donated years ago and sitting around unused. The recovery rate on these kits, which use silica beads and a high pH buffer to dissolve the agarose, are fairly mediocre but good enough. You only need about 25 or 50 ng DNA to perform Sanger sequencing.

Anyway, please look here for a detailed description of my methods and priorities. My goal is to produce very high fidelity reads, which is why I'm doing everything myself and being so anal about the cleanliness of each step.

I really hope you post a full writeup when your results are in. This is a great project, thanks so much for sharing. Also, one more note and them I'm done! The PCR tubes are better placed in the middle of the heat block to ensure consistent thermal cycling.


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Edited by AndyHinton (03/02/20 03:40 PM)

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: AndyHinton]
    #26513079 -

Excellent information thank you yeah the mushrooms I use I am pretty good about extracting inner clean tissue just like I do for clones and then for the CORDY I am using MYC from a dish so I try and scrape it so that I take as little agar with me as possible but I always take some so agar would really be my main contamination. I suppose it could also depend upon the species of mushroom and if they have any compounds within them that inhibit PCR. Yeah Jean was only charges eight dollars for unpurified DNA it’s only two dollars more or something so I just let them do it not that I wouldn’t like to purify my own in the future just because I like doing stuff like that.
I should also have a crisper kit from the oden soon. I have a lot of homework to do but I’m starting to gain momentum knowledge wise. Alan is the primary person teaching me the stuff and he is going to run through this thread also here soon he will be able to decipher more than I and maybe we can come up with some cool ideas


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JOC PAN TEK
CORDYCEPS MILITARIS

EXOTICS
[

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: Jakeoncid419]
    #26513110 -

No worries. I'm only being nitpicky because I want to see you succeed and produce some great data about cordyceps mating. I found that fermenting the mycelium in broth, and then repeatedly centrifuging and washing the pellet with sterile saline, produces large amounts of clean biomass.

Also, do you know about the DIYbio Google Group? It's a pretty decent mailing list for this kind of stuff, including IRL connections. I know of at least one Swiss in this scene who's based near Lausanne.


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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: AndyHinton]
    #26513289 -

Absolutely man that’s half the reason I made this post was so aspects of the procedure can be improved upon. I got my account set up with TF so I Have access to a bunch of different kinds of extract and amp kits They are on the pricey side though


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JOC PAN TEK
CORDYCEPS MILITARIS

EXOTICS
[

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Re: PCR and Electrophoresis for ID and Cordyceps mating type ID [Re: Jakeoncid419]
    #26620564 -

:threadmonitor:


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