Sending plasmids the easy way, paperless!

Some time ago I did a post about the stability of plasmids stored with and without filter paper. Basically, plasmid DNA stored on filter paper has a shorter shelf life than just plasmid in a lil’ plastic bag. As well, the addition of trehalose further increases dried plasmid stability. Thing is, I never actually described HOW to make lil’ plastic bags filled with plasmid. Whoops! Can’t really expect people to adopt a method if there’s no protocol, eh?

Why would you switch over to paperless? If it was good enough for grandma/grandpa, isn’t it good enough for me?? Paperless is:

1. Faster and scalable, if you’re sending one letter or fifty!
2. Less handling, therefore less chance of cross contamination!
3. Longer shelf life!
4. Much easier for the user to recover the DNA!!!

Let me show you how!

I consider myself something of a plasmid distribution aficionado, it makes me happy to send them and hear about how they’ve helped in your lab! It started off with the odd plasmid request here and there, but over time it’s grown to a significant volume of plasmids sent every week. I’ve received dozens of plasmids from researchers and everyone has their own style of sending plasmids, but one common theme is blotting plasmid on filter paper, and I did that for quite some time.

The problem with the filter paper method is that while it’s “good enough” for the odd plasmid request, it doesn’t scale well. You need to cut up the little bits of filter paper (with clean scissors I hope!), arrange them, blot a few uLs of plasmid on, let it dry and cover them with plastic wrap or something. What if you’re preparing a few sets of plasmids? For a few people? One little gust of wind and your little bits of paper are ending up on the floor, start again! Then there’s transferring your now plasmid-laden paper into your pouches, there’s potential for cross contamination. You overcome all that, the poor receiver pays a cool $100 USD for Fedex shipping and they can’t recover plasmid…boo, that’s what we call a feel bad.

So, why not just remove the paper from the equation, and just put our plasmids in plastic pouches? We’ve got two options when it comes to pouches with some pro’s and con’s.

1. Mini ziplock bags
2. Custom heat sealed pouches

I’ll cover #1 first, since it will be accessible to more people. Mini ziplocks, what do they look like? Those with distinguished tastes will appreciate the Apple brand.

Pretty much what you’d expect, 1.25″x 1.25″ polyethylene sacks. A few cents per piece, readily available online or from the dollar store. In terms of sterility you don’t have much to worry about, they’re cranked out by machines in the billions. I know, I know, they’re not RNase/DNase certified but put it this way, a living creature has never touched the inside of one of those.

Let’s prepare the plasmid. If you expect the plasmid to arrive at its destination pretty quickly you can just dilute your plasmid to ~100-500ng/uL with TE. If you want increased shelf life and higher temperature resistance dilute your plasmid to 100-500ng/uL with Plasmid Storage Solution (PSS).

Plasmid Storage Solution (PSS):
40% Trehalose
5 mM Tris pH 8.0
0.1 mM EDTA
Filter, aliqout, freeze

I usually use the above recipe as a 2X solution, such that the final concentration of trehalose is 20%. However, the exact final concentration isn’t critical as long as it’s between 10-40%. Make up your plasmid, diluted with TE or PSS, take 2-5uL and pipette it into your plastic bag. Resist the urge to add over 5uL, dries slower.

See that little drop? That’s your plasmid. You’re basically done! I’d let it dry overnight or in a vacuum dessicator, but it will dry during shipping anyway. If you used PSS the plasmid will be protected in a hard, glassy nugget of trehalose! How do you recover the plasmid? Open the bag, add 50 uL of water or TE, re-suspend and transform 1uL!

Pros to mini-ziplocks? It’s quick! Just pipette a few uLs of plasmid into the bag and seal it up, done! Cons? Well…it looks like a drug bag, a dime bag to be specific. How much of a problem that is depends on where you’re sending from, what country you’re sending to and personal taste. To put it into perspective though, plasmid on filter paper is indistinguishable from LSD blotter. The other con is that you can’t easily remove the air from the bag to improve the shelf life. Want an upgrade? Let’s make some custom vacuum sealing bags!

Equipment Required (?):
1) A vacuum sealer for food
2) An impulse sealer
3) Some vacuum bags

Essentially you’re going to use a combination of the vacuum sealer and impulse sealer to make little pouches of arbitrary size. Here’s a gallery so you can see the progression.

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After you’ve done all that, dilute the plasmids as described above and add 2-5uL to each pouch. Let it dry (or not), vacuum seal it, slap a label on it, done!

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Cons? More effort and equipment. Pros? They look less suspicious and after vacuum sealing the shelf life will increase due to less oxygen and thicker plastic. How to recover the plasmid? Same as above, except this time I can reuse some old stock footage!!!

So that’s how I make the little pouches you get in your packages!

Expanding on the advantages of the paperless method:

1) Fast and scalable. If you send one or two plasmids, just grab a few bags and pipette your plasmids into them, done. No cutting up little papers, sterilizing scissors, transferring the papers, having them blow across the room. If you’re sending 50 plasmids you can make them ahead of time, seal them up and let them sit in your drawer, no problem. Shelf life >1.5 years in a vacuum sealed pouch.

2) Less handling, therefore less chance of cross contamination. You take a fresh tip and aliqout some plasmid into different bags, seal them, no transferring required. With paper you physically have to move the paper to bags or plastic wrap with tweezers or something. More than one type of plasmid? Hope you’re using different tweezers with each one…

3) Longer shelf life. Previously I showed that plasmid in plastic > plasmid on paper in terms of shelf life. Not sure of the exact reason behind the phenomenon. I suspect that filter paper increases the surface area and therefore exposure to oxygen in the air. I also found that trehalose stabilizes dry plasmids, especially when stored at higher temperatures. This can partially be explained by the DNA being trapped inside a glassy matrix of trehalose, again reducing the exposure to the air.

4) Much easier for the user to recover the DNA!!! This is one of the biggest sellers of the method. You literally add some water or TE to the pouch, pipette it up and down, maybe wait a little bit, then transfer it to a new tube. No bits of paper to get in your pipette, no DNA getting left behind in the paper. The plastic pouch is essentially a flat-pack version of a 1.5 mL tube.

General notes on sending plasmids:

1. Do not include anything metallic in your package. One common method I see is filter paper wrapped in tinfoil. Avoid this!! Regardless of the declaration and biosafety letters you put in your package, metallic objects get flagged by postal services and will cause you problems, avoid.

2. Avoid sending 0.2mL tubes and 1.5mL tubes filled with plasmid. Plastic tubes in flat envelopes can burst, leaking plasmid into the letter. I had a plasmid sent to me in a tube that burst…I recovered the plasmid from the paper it soaked into! With the paperless method, you are essentially sending plasmid in a flat tube, much better.