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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Walkthrough of my home lab * 1
    #26604366 - 04/15/20 10:56 PM (3 years, 9 months ago)

Hi guys,

Since everything is closed, I've been shoring up my home lab. I'm sharing it here so people can get inspired to achieve greater things. PCR and agarose gels, Nanopore sequencing, thin-layer chromatography, etc., are theoretically possible. The question is one of reagents and balancing the cost vs. yield of more complex protocols, considering all options.



The lab takes about 15 minutes to set up and take down. The dead air box is a storage bin and the workflow prefers reusable to disposable items, especially for suspicious waste like syringes, needles, petri dishes, chemical containers, etc.

There's ample working space kept sterile by a quality UV-C bulb. I spray things with 70% EtOH, place them in the box, and run the light for 15 min before working. Then I run it between actions whenever there are no biological samples inside.

There's almost 3 ft3 of sterile working space. I typically prop up the edges with Mason jars to make a sash. Shroomery wisdom dictates cutting arm holes, which of course destroys the storage bin.



Extra supplies such as plates and tubes are available with alcohol close by. Eppendorf tubes, pipette tips, and Parafilm backing are the main recyclable waste. A steady hand keeps used tips to a minimum, unless I put it down or change samples.

Consumables ready to disinfect and sterilize. A grounded power strip (obscured behind) for lights, equipment, etc. The power strip also serves ground from a green wire connected at the outlet.



The equipment is modular and changes according to my needs. I prefer small versions of things and adjust my workflow accordingly. Lab equipment is actually quite easy to come by if you're patient and diligent.

Possible modules include thermocycler, electrophoresis, incubator, hotplate/stirrer, sonication, distillation, vacuum pump, microscope, etc. It depends on what equipment you have, what reagents you want to buy, and what standard of excellence you hope to achieve with available options. Some options such as vacuum distillation are very powerful and versatile techniques with little or no consumables dependencies.

A vortex and centrifuge are the bare essentials to shake and stir. A functional lab inventory anchors consumables sets to specific equipment, e.g., to support a microscope you need slides, slip covers, immersion oil, and Kimtech wipes.



In terms of discipline, it straddles a good microbiology lab and a molecular biology one that needs improvement. There's a lot of stuff I'm holding back on because I can do it better and cheaper when society reboots.

That's all. Hope you guys enjoyed it.


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Invisiblebrindle foxx
Doing it my way
I'm a teapot User Gallery


Registered: 11/11/18
Posts: 420 420 Posts!
Re: Walkthrough of my home lab [Re: AndyHinton]
    #26604437 - 04/15/20 11:37 PM (3 years, 9 months ago)

Cool little setup. How often are you using your centrifuge and what practical use
Does it serve besides making PE spores clean for syringes? Also with the vortex are you stirring slants only?


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: Walkthrough of my home lab [Re: brindle foxx] * 1
    #26605019 - 04/16/20 07:14 AM (3 years, 9 months ago)

Thanks. The vortex and centrifuge are for shaking and stirring tubes.

My most recent use of them (yesterday) was to spin down broth into mycelium pellets. I discarded the broth and collected the pellets. Then I and washed them by repeatedly vortexing them with cryo medium, centrifuging them, and discarding the supernatant. Now I have very dense, very clean cryo tubes.

DNA extraction is an example that uses the vortex and centrifuge extensively. You need to mix lysis buffers, shake up ceramic beads, separate out liquids, resuspend pellets in fresh solution, and pass wash solutions and eventually eluted DNA through spin filters.

I wanted to show the logical extreme of compactness vs. capability, where my lab currently is. Using the same setup shown in the OP, it's possible to bring in a thermocycler for DNA/plasmid amplification, a heat block to make GMO bacteria, or a MinION portable sequencer to do Nanopore sequencing.

The question for each equipment module is, "How much would all the reagents cost? Would it be cheaper to wait, send samples for external processing, or do it in house?" For example, I don't want to buy PCR tubes, nuclease-free water, primers, and master mix because they're expensive and I normally have access to those things.

Also, pipettes are much cleaner and less suspicious than syringes. This thread just a visual update, showing you guys some of what I learned since I last had a home lab. :smile:

Edit: For mush cult, the things I've either eliminated or almost eliminated from my workflow include syringes, grains, Parafilm, flames, pasteurization, special lids, and special substrates. I'm simply using Mason jelly jars, with either 25 mL agar or broth, and plastic lids. Then I'm using sterile toothpicks, swabs, and pipette tips to move the mycelium around. Fruiting substrates are either coffee grains (e.g., king tuber truffles), coir/wheat bran (e.g., shaggy mane), or sawdust/wheat bran (e.g., shiitake).


Edited by AndyHinton (04/16/20 07:32 AM)


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Invisiblebrindle foxx
Doing it my way
I'm a teapot User Gallery


Registered: 11/11/18
Posts: 420 420 Posts!
Re: Walkthrough of my home lab [Re: AndyHinton]
    #26605715 - 04/16/20 12:02 PM (3 years, 9 months ago)

Quote:

AndyHinton said:
Thanks. The vortex and centrifuge are for shaking and stirring tubes.

My most recent use of them (yesterday) was to spin down broth into mycelium pellets. I discarded the broth and collected the pellets. Then I and washed them by repeatedly vortexing them with cryo medium, centrifuging them, and discarding the supernatant. Now I have very dense, very clean cryo tubes.

DNA extraction is an example that uses the vortex and centrifuge extensively. You need to mix lysis buffers, shake up ceramic beads, separate out liquids, resuspend pellets in fresh solution, and pass wash solutions and eventually eluted DNA through spin filters.

I wanted to show the logical extreme of compactness vs. capability, where my lab currently is. Using the same setup shown in the OP, it's possible to bring in a thermocycler for DNA/plasmid amplification, a heat block to make GMO bacteria, or a MinION portable sequencer to do Nanopore sequencing.

The question for each equipment module is, "How much would all the reagents cost? Would it be cheaper to wait, send samples for external processing, or do it in house?" For example, I don't want to buy PCR tubes, nuclease-free water, primers, and master mix because they're expensive and I normally have access to those things.

Also, pipettes are much cleaner and less suspicious than syringes. This thread just a visual update, showing you guys some of what I learned since I last had a home lab. :smile:

Edit: For mush cult, the things I've either eliminated or almost eliminated from my workflow include syringes, grains, Parafilm, flames, pasteurization, special lids, and special substrates. I'm simply using Mason jelly jars, with either 25 mL agar or broth, and plastic lids. Then I'm using sterile toothpicks, swabs, and pipette tips to move the mycelium around. Fruiting substrates are either coffee grains (e.g., king tuber truffles), coir/wheat bran (e.g., shaggy mane), or sawdust/wheat bran (e.g., shiitake).




Could you show me the plastic lids you are using to fit those jelly jars and also do you have a good source for sawdust in bulk? And since you are foregoing pasteurization how are you getting your jelly jars clean and how are you getting your shiitake bag logs clean? Are you using just boiling water to make the sub? I’m looking to increase my bio efficiency and also do
Without so many consumables especially non sterile ones like syringes


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: Walkthrough of my home lab [Re: brindle foxx]
    #26606148 - 04/16/20 04:20 PM (3 years, 9 months ago)

The lids are 70 mm regular mouth PP5 with the foam liners removed and a stripe of autoclave tape on top. The autoclave is an All-American stovetop autoclave that's well worth the money. Bulk substrate is Traeger alder fuel pellets.

There are no large spawn bags, horseshit, grain spawn, or any of that. The lab is a portable microbiology/molecular biology lab that's capable of growing mushrooms, not the other way around. If you want bio-efficiency, bottle tek is the way to go. Each container uses about 100g : 30g dry fuel pellets or coco coir : wheat bran, inoculated with about 8 mL broth by pipette.

I thought it'd be cool to post this thread because the lab is totally underwhelming! Not a lot of special stuff, just a tabletop on a bed. But it's a really stripped down example of how you might integrate institutional science into your mushroom setup. For example, you can buy a $30 kit that teaches you how to make fluorescent E. coli from the Odin.

What kind of trickery could result if Shroomery peeps started doing thin-layer chromatography on reishi metabolites, or started designing plasmids to express psilocybin analogues?  The biosynthetic gene cluster for psilocybin is known, I uploaded it to BioTorrents.de. :smile:

Sorry to come off as a telemarketing evangelist. I redid the home lab after neglecting it for 2 years and thought, "If I really needed to, I could sequence whole genomes here."


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Edited by AndyHinton (04/16/20 04:30 PM)


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OfflineThrill
Regnarts
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Folding@home Statistics
Registered: 02/05/11
Posts: 1,928
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Re: Walkthrough of my home lab [Re: AndyHinton]
    #26606868 - 04/16/20 10:03 PM (3 years, 9 months ago)

I understood, like, none of your first 2 posts lol, which means youre just on a whole different level. Makes me feel like s stupid caveman who just learned to grow mold in a bucket.


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Offline24sevenZed
~Z3D3Z~
I'm a teapot


Registered: 11/05/16
Posts: 90
Loc: CYBERSPACE
Last seen: 3 years, 7 months
Re: Walkthrough of my home lab [Re: Thrill]
    #26608787 - 04/17/20 06:44 PM (3 years, 9 months ago)

This is a really useful and informative post, thank you. The correct rabbit hole to be going down I think :cool:

Out of curiosity, have you used MinION and do you think they are reliable? Asking as a layman with no real molecular biology experience.


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: Walkthrough of my home lab [Re: 24sevenZed]
    #26615433 - 04/20/20 01:47 PM (3 years, 9 months ago)

Thanks. I haven't done Nanopore sequencing yet because new flow cells are $900 each and the read quality of washed flow cells is unacceptable. One of BioTorrents.de's funding goals is to split the cost of a flow cell, sequence Chinese "black pearl" oyster's genome, and make it freeleech.

I have the device itself, a very simple thing with USB output. A friend gave it to me along with the tubes needed to shear DNA into about 10 kb fragments. I also have a copy of the pirated black pearl culture within three degrees of separation from the original source.

The MinION seems fairly simple to use. It should give about 95% read accuracy as long as you prepare the sample carefully and don't overuse the flow cell. Other steps in the workflow may give you more trouble, e.g., if you need to concentrate the DNA with ethanol precipitation.

Edit: Some other essential equipment that I may photograph or not. A milligram scale (0.001g) and a small graduated cylinder (0.1 mL increments) to measure things precisely and calibrate your equipment.


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Edited by AndyHinton (04/20/20 01:55 PM)


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: Walkthrough of my home lab [Re: AndyHinton]
    #26641407 - 05/01/20 02:01 PM (3 years, 8 months ago)

Some lessons learned while updating my home lab. I was able to easily accumulate a large amount of essential equipment and consumables in a short time online. There was no need to order anything sketchy, but thin-layer chromatography would have needed hazmats like methanol and caustic oxidizers like potassium permanganate. Anyway, the main things I focused on were measuring, imaging, and validation/error checking, which led me down an accessory spiral.

Pipette tips to transfer liquids to 1 μL and centrifuge tubes to store the liquids. A scale to measure to 1 mg and graduated cylinders to calibrate the pipettes. A calibration weight for the scale itself. A microscope, dSLR adapter, and all the necessary consumables. A stage micrometer to calibrate the images. Autoclave tape and plastic lids to ensure sterility and reduce homemade lid failure. Various electronics to wire up the UV-C bulb. Trying to find cheap alcohol during a plague (impossible, so I use the cylinders to make 70% Everclear in dH2O).

This all cost money but now I can take uncommonly accurate measurements for a hobbyist. The ability is intrinsic to the lab because I own everything necessary to validate a set of equipment for basic physical measurements (size, volume, mass). But there's always more to buy, unfortunately. Looking back, I would have prioritized equipment that requires no extra stuff to work, such as an infrared digital thermometer.

Hope this helps. I've found that you can ape a lot of "real" lab technique with common recyclables and simple mods to used equipment, which is often cheap or free if you know where to look. I wouldn't take shortcuts with the quality of the equipment or the reagents. Use Sci-Hub often, gradually build a sustainable setup, and soon enough the forefront is right there.


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OfflineSolipsis
m̶a̶d̶ disappointed scientist
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Registered: 12/28/09
Posts: 3,398
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Last seen: 5 months, 18 days
Re: Walkthrough of my home lab [Re: AndyHinton]
    #26642061 - 05/01/20 07:34 PM (3 years, 8 months ago)

Very nice buddy, thnx for sharing <3
inspiring


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OfflineAz88
Stranger
Registered: 05/04/20
Posts: 88
Loc: Texas Flag
Last seen: 3 years, 1 month
Re: Walkthrough of my home lab [Re: Solipsis]
    #26647867 - 05/04/20 09:32 AM (3 years, 8 months ago)

Congrats!! I have a Gorilla Kit set up and love it personally.


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