Home cloning setup

[Michael Flynn]

Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

[Skyler Gordon]

You can probably skip the -80C for a while, not really necessary unless you want to keep bacteria from mutating or you have some RNA you want to keep for a long time.

-SG

On Fri, Feb 23, 2018 at 9:32 PM Michael Flynn <mfly...@gmail.com> wrote:

Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

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[Koeng]

Hi Flynn, 

I actually do have a cloning setup at home. My lab is actually specifically made for cloning (I like to figure out how to clone things well while working on the Free Genes project, then implement those things at home). And no, I'm not rich. I rent a single small bedroom, which is both my lab space and my home. 

Incubators are cheap from ebay. ~$100. I put an orbital shaker I got for $50 into it, so now I have both a shaking and stationary incubator. You also don't really need an incubator... bacteria grow just fine at room temperature, albeit for longer times. I often grow my sporulating Bacillus at room temperature so they don't take up room in my incubator. 

I don't actually have any freezer room except a small box for enzymes that I keep in our main freezer that I share with roommates. Unfortunately, that also means I don't really have space to store any food, so I usually keep dry food at home for eating. If I had room, I'd pick up a $200 chest freezer from Home Depot, that could store all my stuff for the foreseeable future. Oh, the glories of having space...

I don't have a -80C freezer. Those things are expensive man! And large. Right now I just use competent cells as I make them. That is getting very annoying, so I plan on paying for storage of my competent cells at a local biohacking lab. @Sebastian has also reported that the cells stay good for a few weeks at -20C, so it is quite possible if I had a chest freezer I could just make new batches every other week. 

For long term storage options, I've found that cells last for about a year at -20c in glycerol. They start doing funky stuff after that, so if you regenerate stocks every 6 months, it should be fine. Or just store plasmid stocks. Another good method is to spin down the cells just like you are about to do a miniprep, then freeze those stocks. If you need the plasmid, go ahead and do a miniprep of that frozen stock. It works well after a couple of months, and I haven't tried a yearlong experiment yet. 

You don't need a hood. Why would you need a hood? You don't even need a bunsen burner! Pick up an air filter from amazon for about $150, and that will be MORE than enough. In addition, when you make LB and the dust gets everywhere, the air filter will make sure your room doesn't get all dusty. It also keeps things nice and sterile, just like a bunsen burner. To spread cells, just keep a bic lighter handy and a small beaker of ethanol (from CVS). Dip your inoculating loop in the ethanol, then just do a quick light to burn that ethanol off. Keeps it sterile. 

I use GoldenGate enzymes. No need for PCR reagents. I have 5 enzymes - T4 Ligase, T4 PNK, SapI, BbsI, and BsaI. About $250 off of NEB. I don't like PCR for several reasons. However, if it fits your fancy you can get those enzymes for a couple hundred more bucks.

I don't need microscopes. Why would I need a microscope? Why would I need a FEW microscopes? Here's a tip - learn what your bacteria smell like. I recall one time I caught contamination of my yeast stocks because I took a whiff of the overnight and it didn't have that familiar baker's-yeast smell. Bacillus subtilis is quite easy because it smells so much like dirty feet. E.coli also has a certain smell. You have a great biochemical sensor built into yourself! Use it!

List:

Enzymes : $250

Incubator + shaker: $150

Centrifuge : $100

PCR Machine: $150

Air filter: $150

Glassware: $100

Pipettes: $150

Disposables: $100

Media: $100

Other expenses (tables, boxes, tube holders, etc etc. Everything else you can think of): $250

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Total: $1500

That seems expensive, but remember I have a very complete lab that I am quite proud of, and probably spent around the range of $2500 on over the range of the last 5 years (high-throughput is expensive ): ). If you're clever, I bet you can cut costs but many hundreds of dollars, and get a cloning setup to do your specific experiment for about $500. If you need any advice, hit me up. But no, you don't have to be rich. I'm certainly not rich, just dedicated :)

If you're not sure how to get all that stuff, Josiah has made it quite easy at the-odin http://www.the-odin.com/genetic-engineering-home-lab-kit/ 

Otherwise, back to eBay!

Koeng

[djwr...@gmail.com]

Hi Koeng,

What kind of air filter do you use? Do you have to change it after a certain amount of time?  We have some competent cells you can use. 

Thanks,

Dan

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[Koeng]

I use the honeywell air purifier - https://www.amazon.com/Honeywell-50250-S-True-HEPA-Purifier/dp/B00007E7RY/ref=sr_1_5?s=home-garden&ie=UTF8&qid=1519684656&sr=1-5&keywords=Honeywell+air+purifier (this one is larger than the one I have, though I couldn't find the one that I do. Must be discontinued)

I probably need to change it, but I haven't had to thus far. It is a bit better than what I need, since I have a small room. 

Awesome :) will ask to ship some soonish

Koeng

[Steven Harlow]

Hi Koeng,

That's a really cool setup! Impressive that you're able to make good use of such tight space. Do you mind if I ask what you do most of your cloning for?

Cheers,

Steve

[Tres Brazell]

Quick question about your GoldenGate enzymes.  Did you order those from NEB? Was it hard to set up an account with those folks? Thanks for the post btw!

On Monday, February 26, 2018 at 11:07:32 AM UTC-8, Koeng wrote:

[Koeng]

@Steven I'm mainly actually cloning for the sake of cloning, so mainly doing optimizations and figuring out how to most efficiently make DNA. Right now, I'm working on a DNA synthesis method using restriction enzymes.

@Tres Yes, everything from NEB. They won't send to a home address, you need a business address. Other than that, they're great. I trust them with all my enzyme stuff. Very reliable. 

Koeng