Getting new plasmids in the mail is like Christmas, there’s nothing like the anticipation of cracking open a letter and adding a new enzyme or technique to your toolbox. The logistics of non-profit plasmid distribution can be tedious at times, but getting feedback about how your labs made buckets of enzymes and saved thousands of dollars make my day 🙂
Now, I ain’t Addgene level of fancy, but I do try to keep things classy. I always thought that the classic “plasmid on filter paper” method was adequate for sharing plasmids. Just like nonna used to make! I even vacuum pack them to reduce the chance for cross contamination. The question always stuck with me though, how long does DNA last on paper, and is there a better way to do things?
I finally cracked the seal on a 1.5 year time trail of plasmid DNA on paper and the results are fascinating! Not only that, but I think we have a far superior way of sending plasmids now!
In the distant mists of time, some would say 1.5 years ago, my past self thought it would be a cool idea to leave plasmid blotted on filter paper strewn about the lab. I blotted about 2ug of pfu-sso7d onto little pieces of filter paper, let them dry and vaccuum sealed them. Then they were put in various locations in the lab. Looking back I should have started all the samples on the same day, done replicates, more standardized blotting, etc etc. Forgive Past Alex, he was doing it for kicks, not realizing that one day readers would appreciate these details. Still, interesting stuff to be seen.
I put the plasmids at room temperature (RT), RT in a dessicator (RT+D), 4c fridge, -20C freezer, in the 37C incubator and finally in front of a sunny window (RT+Sun). I also found some other plasmids that never got sent out, including a few where I tried putting the plasmid into the pouches without paper, something I was playing around with at the time.
I took a 4mm disc from each filter paper, soaked it in warm Tris pH 8.0 for 5 minutes and transformed 100 uL of DH5a with 5 uL of eluted plasmid. For the plastic pouches I added 50 uL of the warm Tris to the bag, pipetted up and down a few times, and let it sit for 5 minutes, transformed 5 uL into 100 uL cells. Let’s take a look at the plates, first the RT (24/7/18), RT+D (24/7/18) , 27/3/19 and 15/5/19 samples.
Yeah…damn. Looks like even 5 months on filter paper is enough to seriously affect the amount of colonies retrieved from the paper. It’s true, you only need one colony, but not what I’d like to see from 2ug input. How about more extreme temperatures?
Very interesting! You can significantly extend the life of plasmids on paper by putting it at a low temperature. Someone sent you a plasmid on paper? Throw it in the fridge. No surprises with the 37C incubator and sun, no transformation except for the single colony on the sun plate, it’s a survivor (or cross contamination), I respect that. What about those paperless pouches you ask???
That’s more like it! I think that picture speaks for itself, the paperless method absolutely crushes the classic method. The interesting question is why? Reduced surface area? Less contact with air? Is the paper corrosive? It’s sterile, I autoclaved it, so I don’t think it’s being degraded that way…
Either way, considering that 5 months later a pouch of plasmid can give you a lawn of bacteria, this will be the preferred method of sharing plasmids going forward.
Still working on making this method a bit more user friendly.
Paperless Plasmid Pouch (PPP) Method V1.0
1) Add a few uLs of purified plasmid into vacuum sealing pouch. Anywhere between 500ng and 2ugs seems like plenty.
2) Open the pouch so the water can evaporate, the fewer uL you put in the pouch the better. Dry in a dessicator or a centrivap if you have one.
3) Vacuum seal the pouch for the sake of fanciness
Tools/Materials I use for this setup, you can make arbitrary shaped and size pouches:
– Vacuum heat sealer for food (Amazon Affiliate Link) (?)
– Extra heat sealing bags (Amazon Affiliate Link) (?)
– Impulse sealer (Amazon Affiliate Link) (?)
Here’s a visual guide of how to the retrieve the plasmid:
What kind of pouch is it? Brand / make? Do you sterilize it some way?
I’ll put a description and links up in the post, but the pouches are made of the same vacuum sealing bags I used before for packaging the filter paper. I then vacuum seal after the plasmid is dry. I’m also experimenting with the addition of 5% trehalose for further stability.
Nope, no sterilizing, I figure no human has laid hands on the insides of the plastic bags. Obviously not gamma sterilized…but you can’t have everything in life
The way you stored dried plasmids in the pouch is amazing. I am just wondering about plasmid supercoied form between the one stored on paper at -20, room temperature and the paperless pouch at room temperature.
How did the 5% trehalose work out?
Trehalose worked awesome actually! It greatly extends the shelf life of dried plasmids, It’s standard for me now when sending out plasmids(10-20% final concentration). Dries out into this nugget that protects everything in a glassy state and it’s easy to re-suspend.