The goal here is to achieve a nice even suspension of each cell line so they will spread out evenly. Avoid excessive jarring of recently tryplated colonies as this will tend to lump the cells to one side of a well.

-On the second day after picking colonies aspirate the medium and wash once with 180 µL of warm PBS. When adding solutions to wells squirt against the side of each well, that way you won't need new tips for each row. (the chunks of colonies will often be distributed to one side of the wells. If so you can turn the plate around so that the aspirator touches the empty side of each well).

-Apply 50 µL of recently thawed trypsin (prefer fresh since this is an important step) and put the plates in the incubator for five minutes. (the trypsin will be stopped with medium next step–thus you can do up to 5 plates).

-Observe that the chunks of colonies are floating nicely and add 180 µL fresh medium with G418. Do this to every plate to stop the trypsinization.

-Set the octapipetteman to 150 µL (to avoid sucking up solution into the barrel). Draw each row up vigorously 5-8 times until the suspension are single cells. Change tips each time for each row.

-Observe how well broken up they are after doing a few columns to evaluate your technique. If still not single cells then try more pipetting. If that doesn't work (if for instance the trypsin was bad) then do the best that you can. You'll want to repeat this whole procedure tomorrow after they've attached, rather than allowing a large proportion of your colonies to grow as clumps–checking early will make this highly unlikely.

-Place the plates back in the incubator in an out-of-the-way spot. Try not to jar the plates much as this will tend to lump the cells to one side rather than settling down evenly across each well.

-Promptly change the medium the next day, preferably early the next morning so that they don't sit in diluted medium for a long time. Change medium daily hereafter for 3-5 days until they are highly confluent. Maintain G418 throughout these steps to weed out any false positives and assure clonality to your lines.