Pinning from 96-well plates
Materials and Reagents:
- Frozen stocks:
Available through Research
YPD + 2% agar + G418 (concentration of 150µg/ml)
- Pinning tools:
Long pin versions are available from V&P
Scientific. They have a large assortment of pinning tools with
different characteristics.The one that I have is model VP408B, but
there are many to choose from. If you mention the pin tool is for
use with the Sacchromyces Genome Deletion Project, they will
give you a 5% discount.
Another alternative for a simple 96 well pin tool is: Denville
Scientific Inc., item number P-8850.
- Plate sealers:
- There are many versions of these and can be found in most lab
I use the Thermowell sealing tapes from Corning/Costar.
1. Thaw the frozen plates. They come as frozen stocks in YPD + 15% glycerol.
It is easier to peel off the foil coverings if the plates are still
Let the plate thaw completely on a flat surface.
2. Gently stir the liquid glycerol stock with the pinning tool and
plate onto YPD + 2% agar + G418 using a 96 well pin tool.
Make sure the pins don't drip liquid into neighboring wells.
The agar can be in any format that can accommodate the pin tool:
50 x 15mm petri dishes
96 well microtiter plates
3. Make a duplicate plate by pinning the culture into a 96-well microtiter
filled with YPD.
Leave room for the freezing agent.
This is an optional step; this plate is for a backup freezer stock.
4. Clean the pin tool between plates!
a. Dip the pin tool in a deionized water to dislodge the yeast. Then
dip into 95-100% ethanol and flame the pin tool. Make sure the pins
are cool before placing it into the next plate since the hot pins
will kill the culture.
b. Wash the pin tool first in deionized water, then 70% ethanol. Blot
off the moisture, then wash in acetone and air dry. (ref: Sharon Sookhai-Mahadeo)
5. Grow the plates for 2 days at 30°C without shaking.
6. Add glycerol (final concentration to 15%) or DMSO (final concentration
to 7%) to the liquid cultures. Seal well and freeze.
7. Reseal the stock plates with fresh plate sealers and refreeze.
We don't recommend pinning from a frozen plate since the ice chips
may contaminate neighboring wells.
We have no preference to what format the YPD + agar (i.e., plates
versus individual microtiter wells) is in. We have used all the methods
satisfactorily. It's a matter of personal preference and what you
plan to use the strains for.
So far we have refrozen our stocks about three times and haven't
seen a decrease in cell density.
This is one suggestion for how to process them. If you have another
protocol that you'd like to offer, please send comments or comments