Colonies are usually picked at about day 10 of neo selection (day when neo is first applied is counted as day 0). Aspirate media, invert plate, and use a colored marker to mark colonies under a stereoscope. Care should be taken to identify undifferentiated colonies, and different colored markers can be used to identify clones in different pools. Cover the plate with 10 ml of PBS. Using fine drawn capillaries, pick about one fifth of each colony, and repeat this process with the same capillary for the remaining colonies in the pool (from 5-50 colonies). Although the same capillary is used throughout this process, we have found cross-contamination between colonies not to be a problem. Transfer picked colonies to a 0.6 ml Eppendorf tube. Spin cells down 30 sec. and resuspend pellet in 12μl PCR lysis buffer (1x Thermopol buffer, 50 μg/ml Proteinase K, 1.7 μM SDS).
Once a PCR positive pool has been identified, or if individual colonies are to be picked, proceed as described above. At this point, we use different capillaries for each colony. If care is taken, it is also possible to pick just a little from each colony and transfer cells directly to a tube containing 12μl of PCR lysis buffer with minimal PBS carry-over. This way, it is not necessary to spin cells down and resuspend.
The PCR assay is done as follows:
-Lyse cells in PCR lysis buffer for 60 min. at 55°. Denature Proteinase K for 10 min. at 85°. Spin down.
-PCR reactions are done in 25μl final volume as described in our PCR protocol. You can make a master mix with everything except DNA.
-Dispense 20μl of this mix per tube prior to adding 5 μl DNA, then overlay with light mineral oil. Oligos originate from exogenous sequences in the targeting vector (e.g. neo gene) and sequences outside of the targeting vector and are designed using the “Primer 3” program or MacVector, usually 20mers, about 50% GC. Melting temp. is usually 55°-60°.
PCR conditions are optimized using a mock construct (containing sequences recognized by both oligos) diluted with genomic DNA. Test PCRs are done in 25μl final volume, using 100 ng of genomic DNA (e.g. tail DNA), and dilutions of mock plasmid. We usually try to get a UV visible product after 40 cycles using 1 fg of plasmid DNA, and for an extension of 2-3 kb. PCR extension times are based on 700 bp/min. If the PCR product is over 2 Kb, add Taq Extender to the reaction.