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Picking Colonies

At this stage you should have planned and optimized your screening strategy.  There are several options. On this website are protocols for a mini-Southern, PCR from colony picks, and a full Southern.

Picking colonies occurs around 8-10 days after ES colonies have been cultured in drug selection media.  A 96-well feeder plate is usually used to grow DNA for mini-Southern or a 96-well v-bottom plate for PCR analysis. The strategy for splitting some of the picked colony onto a 96-well plate for PCR or mini-Southern analysis is to allow you to screen your colonies and to only continue to expand your positive clones from the 24-well master plates. You may also choose to pick into two 96-well plates, one for a master plate to freeze down, and a second for analysis.  Later you can thaw and expand your positive clones from the 96-well master plate.  If you do not get clear results of which clones are positive it is best to continue to expand and freeze down all of your clones and do a Southern. 

Also you must decide whether to pull drugs from the colonies or not.  Our policy is to continue using drugs. Although the cells will grow at a slightly slower rate and you will loose some colonies in the initial split, this is not a concern since the colonies lost were probably mostly wild-type cells.  Continuing using drugs will also help get rid of any wild-type contamination from mixed colonies.

1.   Prepare feeder plates the day before (one 96-well plate together with one 96- or 24-well master plate). On average most people pick 200-300 colonies.  There should be more than enough colonies to pick from your 10-cm plates.
        The master plate you will need is based on your screening strategy (whether you are doing PCR on picked colonies, mini-Southern, or direct genomic Southern).  You do not need to prepare the first 96-well feeder plates if you are planning to do an immediate PCR screen from your colony picks.  Have v-bottom 96-well plates on hand and use in place of the feeder 96-well plate. 
2.  Feed the cells 2 hours before picking and change the media on your 96- and 24-well plates to fresh ES cell media.
3.  Prepare a 96-well plate with 30ul of trypsin in each well.
4.  Wash a 10-cm plate with PBS and aspirate. Add another 10mls of PBS.
5.  Pick 8 colonies with a mouth pipette or a pipetteman set at 10 ul.  Look for robust colonies that have defined edges.  Do not pick colonies that are very large and flat, these are most likely undergoing differentiation.  It is best to do this as quickly as possible.
6.  Incubate the plate at 37° C for 5 min.  At this time you can begin picking anther set of 8 colonies in another prepared 96-well plate with trypsin.
7.  After 5 min, add 60 ul of ES cell media to the first set, titrate, and transfer 50 ul to the 96-well plate (feeder plate or v-bottom) and 80 ul to the 24-well or 96-well master plate.
8. When you are finished picking colonies from one10-cm plate replace PBS with ES cell media and place back into the incubator.
9.  Repeat with next 10-cm plate.
10.You can culture the plate and pick more colonies the next day if desired.  Soon the colonies will begin to differentiate.  Discard the10-cm plates at this point. 
11. Continue to feed your 96-well and 24-well plates as needed. 



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