Not sure about the best way to store your plasmids long term?
I’m Racheal, a lab tech at Addgene, and in this video, we’re going to show you the
most effective way to store your plasmids indefinitely:
by Creating Bacterial Glycerol stocks.
Bacterial glycerol stocks are incredibly important,
if you’re planning on storing plasmids for an extended period of time.
This way, when you want to make more plasmid DNA, the plasmid will already be in your desired
bacterial strain and you will not need to obtain more competent cells and retransform.
Bacteria on an LB agar plate can normally be stored for a few weeks in the fridge.
Storing the bacteria in a tube and adding glycerol before freezing will stabilize the
bacteria, preventing damage to the cell membrane and keeping cells alive.
This allows you to store a glycerol stock at -80°C for many years.
To get started, you’ll need to grow your cultures overnight.
If you need help, you can follow our steps for Inoculating an Overnight Liquid Culture,
or watch our video.
Before continuing, you’ll need a few materials:
A 2 mL screw cap or cryovial
50% glycerol
Liquid Culture with Bacterial Growth
1000uL pipette and tips
70% isopropanol
Sterile loop or autoclaved toothpicks
Dry Ice
And a LB Agar Plate
First, you’ll need to prepare your 50% glycerol solution.
You can do this by diluting 100% glycerol in dH20.
Label the tubes by using a marker and writing directly onto the tube.
It is important that you do this before freezing, because frozen tubes are difficult to write on,
and labels can easily unstick from tubes at low temperatures.
Snap top tubes are not recommended, because they can open unexpectedly at very cold temperatures.
Once you have bacterial culture and your diluted glycerol solution, add 500 μL 50% glycerol
and culture to 500 μL of the overnight to a 2 mL screw top tube, or cryovial.
This gives you a final glycerol concentration of 25% for your glycerol stock.
Shake the tube five to six times to thoroughly mix the glycerol with the bacterial culture.
Once there are no layers present and you see one uniform solution, the tube is ready to
be frozen.
Place the glycerol stock tube in a freezer at -80°C. Congratulations!
Your stock is now stable for years, as long as it is kept at -80°C.
Keep in mind that multiple freeze and thaw cycles will greatly reduce the shelf life
of your stock.
To recover your bacteria, remove the stock tube from the freezer, and open it.
Then, use a toothpick, sterile loop or pipette tip to scrape some of the frozen bacteria
off the top.
Do not let the glycerol stock thaw!
Place it on dry ice while streaking to prevent the stock from completely
thawing, preserving its shelf life.
Now, streak the bacteria onto an LB agar plate.
If you need help, check out our text protocol and video on streaking bacteria.
Grow your bacteria overnight at the appropriate temperature.
You can check growth conditions, including copy number and growth temperature, on your
plasmid’s information page.
The next day you will be able to start an overnight culture for plasmid DNA prep.
Thanks for watching our protocol video on Creating Bacterial Glycerol Stocks for Long-term
Storage of Plasmids.
Leave a comment below to let us know what videos you’d like to see in the future,
or to tell us how we can improve.
Check out our other protocol videos, like Inoculating an Overnight Liquid Culture and
Streaking and Isolating Bacteria on an LB Agar Plate here on our Youtube channel, or
head to addgene.org/protocols for a full list of our written protocols.
Addgene: a Better Way to Share Science