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Protocols: ES Cell Culture - Gene Targeting - Protocols

Analysis of ES Cell Clones by Mini-Southern

Initial Cloning

	1.	Aspirate selective media off the plates containing colonies of 
interest.  Wash the plates twice with PBS.  After aspirating the second wash, 
add enough PBS to cover the surface of the dish.

	2.	Prepare the 96-well U-bottom cloning dish by pipetting 25 ul 
of trypsin into each well using a multichannel pipettor.  Up to two full 
plates of clones can be picked at one sitting without problems of the 
trypsin drying.

	3.	Pick colonies from the washed plates and transfer them into 
the trypsin solution, one colony per well.  Proceed until colonies have 
been cloned into each of the 96 wells.  Approximately one hour will have 
elapsed by the end of the process (assuming a picking speed of 100-150 
colonies/hour), enough time to ensure complete trypsinization of the 
colonies.  Therefore, no further incubation is necessary.

	4.	Using the multichannel pipettor, add 25 ul of M15 media 
per well.  Pipette up-and-down several times to disaggregate the cells.

	5.	Transfer the clones to an appropriately labelled 96-well 
feeder plate and allow the colonies to grow under selection.  The cells 
will take about 3-5 days to grow to such a confluence as to turn the 
media yellow at one-day intervals.

Duplicate Preparation

	1.	Once the clones reach confluence, trypsinize, duplicate, 
and freeze the cells [refer to the protocol "Freezing Embryonic Stem 
ES) Cell Clones in 96-Well Plates"].

	2.	Allow the cells to grow on the gelatinized 96-well 
duplicate plates.  Refeed the plates with fresh M15 media as the old 
media turns yellow until the cells are ready for use.

Mini-Southern Analysis

	1.	When the cells are ready, wash the plates twice with PBS 
and aspirate.  Using the multichannel pipettor, add 50 ul of Lysis 
Buffer [10 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 10 mM NaCl, 0.5% Sarcosyl, 
and 1 mg/ml Proteinase K (added fresh)] per well.  Note:  From this 
point on, all manipulations are to be performed outside the tissue 
culture facilities.

	2.	Incubate the plates overnight @ 60o C in a humidified 
chamber (such as a plastic container with a wet sponge on the inside).

	3.	Next day, prepare a fresh solution of 75 mM NaCl in 
ethanol (add 150 ul of 5 M NaCl per 10 ml of cold absolute ethanol 
and mix well; the salt will precipitate, but this is of little consequence).

	4.	Using the multichannel pipettor, add 100 ul of the 
NaCl/ethanol solution per well.  Allow the plate to rest on the bench 
@ room temperature for 15-30 minutes, or until the precipitated DNA 
is clearly visible under low-power magnification.  The DNA adheres to 
the plastic, so look at the perimeter of each well to see the 
precipitated DNA.

	5.	Invert the plate to discard the solution (the DNA will 
remain adhered to the plate).  Using the multichannel pipettor, 
add 70% ethanol to wash each well.  Alternatively, a squirt bottle 
may be used, but a strong stream could detach the DNA from the plate.  
Invert the plate to discard the 70% ethanol and repeat the wash 2-3 
times.

	6.	After the final wash, invert the plate, discard the 70% 
ethanol, and allow the plate to air-dry a few minutes.

	7.	While the plate is drying, prepare the Restriction 
Enzyme Cocktail (1X Restriction Buffer specified for the enzyme 
being used, 1 mM Spermidine, 100 ug/ml Bovine Serum Albumin, and 
10-15 units of enzyme).

	8.	Using the multichannel pipettor, add 30 ul of Restriction 
Enzyme Cocktail to each well and mix by pipetting up-and-down.  
Change tips between one row and the next.

	9.	Once the cocktail has been added to all the wells, 
incubate the plates overnight @ 37o C in a humidified chamber.

	10.	Next day, prepare the agarose gel(s) for electrophoresis.  
Prepare a large gel tray (15 cm x 24 cm) with three 33-teeth combs 
(add 3 extra teeth per comb, secured with tape) evenly distributed 
along the length of the tray.  Pour 300 ml of molten agarose into the 
tray; remove any bubbles with a needle.  Allow the gel to solidify 
for about 30 minutes.  This size of gel (3 lanes with 99 wells total) 
will accommodate one 96-well mini-Southern digest plate at 32 samples 
per lane plus one well per lane for molecular weight markers.

	11.	Remove the 96-well mini-Southern digest plate from 
the incubator and add 4-5 ul of loading buffer to each well.  
Load the gel (30-35 ul per well) and run @ 80 V for approximately 
4 hours.  When deciding how far to allow the samples to migrate, 
take into account the size of the fragment(s) being distinguished.  
The gel can be run further as long as the bands from one lane of 
samples being detected with a probe do not overlap the bands 
from the next lane of samples.

	12.	After the electrophoresis is complete, transfer the 
DNA to GeneScreen Plus membranes according to the usual protocol 
[refer to the protocol "Genomic DNA: Restriction Enzyme Digests, 
Agarose Gel Electrophoresis, and Southern Transfer (Blotting)"].

NOTES:	This protocol may not work efficiently for all 
restriction enzymes.  Therefore, make a pilot experiment to test 
the desired enzyme(s) before proceeding to the large experiment.

	Before transferring the DNA to membranes, discard the areas 
of the agarose gel that are not needed for probing.