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Pinning from 96-well plates


Materials and Reagents:

Frozen stocks:
Available through Research Genetics, ATCC, and EUROSCARF.
 
Media:
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 equipment catalogs.
I use the Thermowell ™ sealing tapes from Corning/Costar.
 

Protocol:

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 frozen solid.
    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
       Omni Trays
       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 30C 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.

Additional comments:

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 to: amchu@cmgm.stanford.edu