David LePage's Gene Targeting Protocols

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Thawing Lines From 96-Well Plates

Once you've accumulated some long exposures to be sure of the entire plate, get the positives out of the 96-well freeze ASAP. After thawing them you'll quickly see why I said this was a high-stress freeze. You can count on being miserable during this anxious period: remember they usually pull through.

-As with any thaw you'll want to do this quickly. The fastest way I've found is to stack old yellow pipet tip racks in the 37 degree water bath until there is only a few millimeters of warm water above the rack (obviously you don't want the water to be so high that it seeps into the lid--practice with an old plate).

-Remove a frozen 96-well plate (keep any others in the box at -80) and remove the parafilm. Put the plate on the rack in the 37 degree water bath. To keep it in close contact with the water put a lead pig or spare beaker on top to weigh it down. Usually takes 8 to 10 minutes to thaw.

-Once you can see that the majority of clones have thawed (the wells will be translucent) remove the dish from the water. Place on top of 70% ETOH soaked kim-wipes and wipe all around the top and edges.

-Clearly mark all your positive clones. Also select one or two non-targeted lines for negative controls.

-Using a P200 set at 105 µl, stab under the oil and gently stir the well to mix. Draw up the entire volume plus a little oil. Place this in a 24-well with 0.5ml of warm culture medium. Rinse the tip to disperse cells evenly and remove 105 µl of the 24-well and put it back under the oil of the positive clone. Draw up repeatedly to mix and get most of the cells. Return this to the 24-well. Repeat--you can also examine the 96-well to see that most of the sample has been removed. Do not be concerned with little bits of oil--they can easily be skimmed off the 24-well later.

-Allow to grow overnight and replace with fresh culture medium promptly. At the earliest this can be done about 12 hours after plating. I usually remove and hold onto the 0.5 ml from each well until I'm sure there are cells attached to the 24-well.

-Since they are unhappy I refrain from all drugs. I feel that once picked and grown up for Southerns in the presence of G418 then a line is clonal and does not need any more selection.

-They will usually be refractory and look awful for a day or two. If the 24-well is vigorously growing, then you can passage to a fresh 35 mm dish (that's a 1:5 split). If still looking poorly, passage to one or two fresh 24-wells. Sometimes this passaging gets them jump-started. Don't leave them on the original 24 well for more than four days (feeding daily of course). Once up to the level of a 35 mm dish you can passage them normally and freeze a vial or two back. Some derivatized lines act a little different in terms of growth dynamics--as always be flexible and use your best judgement.

-In addition to preparing a plate or two for freezing I also prepare 1 x 35 mm for DNA. This is overkill but ensures a ton of DNA for additional characterization. It also requires a different protocol (see below).

-I also prepare 1 x 35 mm for karyotyping, but stop at the first fixative step (step #7, below), leaving them in the fridge until I'm sure they are targeted and worth preparing spreads. This way I can leave my founder vials undisturbed until it is time to inject. When just starting out I wouldn't bother: instead relax and bask in the pure joy of having your lines safely frozen back.


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