David LePage's Gene Targeting Protocols

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Freezing In 96-Well Plates

Realize from the outset that this is a high-stress freeze and colonies are not happy. Have all your probes already figured out and reagents ready for action so that you can retrieve your colonies from freezing as quickly as possible. Only one diagnostic southern is required to reduce the number of colonies to a manageable number–analyzing the other side of the recombination and confirmation with additional probes can wait until you have thawed your lines and know that they look OK.

-There are several items to prepare ahead on the day of freezing:

Feed the highly confluent plates 2-4 hours in advance (no drugs).

Gelatinize 2 x 96-well plates for each original plate. These are for making DNA replicates.

1 x 96-well freezing plate for each original plate:

U-bottom polypropylene 96-well plates and matt caps from Marsh Bio Products; autoclaved. To each well is added 30 µl of 2 x freezing medium (This is plain Iscove's containing 20% serum and 20% DMSO (hybri-max)–freshly made and kept on ice). Overlay each well with 100 µl of sterile mineral oil (embryo tested, 0.2 micron filtered). Put the labeled matt cap on loosely and place the plate on ice in dark.

Sufficient medium to put 105 µl/well.

-When everything is ready, wash each plate once with PBS and apply 25 µl of fresh trypsin.

-Place the plates in the incubator for 5-10 min. Gently agitate the plates and ascertain that the colonies are floating freely.

-Apply 25 µl of medium per well. At this point you can divide the plates into sub groups of 2 to process for freezing.

-Put the appropriate labeled freezing plates on a shallow tray with flat compacted ice.

-Set the P50 octapette at 30µl and vigorously break up a column of colonies. Transfer 30µl to the appropriate column of the freezing plate, stabbing under the oil and mixing gently 3 times.

-Repeat for each column using fresh tips until finished. Fully place the matt cap on the freezing plate and return it to ice. Process the second plate.

-When both freezing plates are finished transfer them to a stryrofoam box (NEB shipper or a pair of lids to a Sarstedt freezer tray taped together) and place the box in the -80.

-Repeat the processing steps until all the plates are in the -80.

-Now return to the original plates (which have 20 µl/well remaining) and add 80 µl/well. Return them to the incubator temporarily.

-Put approximately 100 µl/well of medium in the 2 x 96-well DNA replicate plates. Put these in the incubator as well.

-Remove an original plate and its 2 x 96-well replicates (Do not attempt to do more than one original plate at a time).

-Gently mix the original plate and transfer 50 µl of suspension to each replicate, mixing gently each time. The replicates should now have all the cells.

-Return the replicates to the incubator. Discard the original.

-The DNA plates can usually be ignored the next day, fed the second day and every day thereafter (no drugs are necessary because any false positives have already died).

-After the freezing plates have equilibrated at -80 for at least 48 hours, they can be transferred to liquid nitrogen. Wrap each plate in tin foil (so that the lid can't float off) and put on dry ice. Place two plates into a large dewar box and return to dewar (will be a tight fit but just jam it in there).

96-well Polypropylene U-bottom microplates

(Check autoclaved reserves)

Marsh Bio Products




Autoclavable matt cap for deep well plates (Check autoclaved reserves)

Marsh Bio Products




Mineral oil, embryo tested (Check 0.2 µ filtered supply)



500 ml


DMSO, tissue culture tested, Hybri-max






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