Editing AAV-Production-293T-2 (section)

=== (B) Picking drug-resistant clones: PSCs â TIMING 1 h  ===

<br> (i) Seed a 24-well plate with 3.7 Ã 104 irradiated MEFs per well, 1 d before picking resistant colonies. Place in a 37 °C incubator overnight. 

(ii) The next day, wash the 24-well plate with 1Ã PBS and add 0.5 ml of ESC medium. 

(iii) Identify drug-resistant colonies by scanning the dishes (from Step 56B(xxviii)) with an inverted microscope and circle the resistant colonies underneath the dish with a permanent marker. 

(iv) Prepare calibrated pipettes by pulling them over a flame to refine the tip. The new ends should be shaped in the form of a small needle with an opening. 

(v) Set up a stereomicroscope in the tissue culture hood. It can be wiped with a 70% (vol/vol) ethanol solution to improve sterility. 

(vi) Assemble the mouth pipette by attaching rubber tubing on either side of a Millex GS filter unit (0.22 ÎŒm). Attach the small red mouthpiece to one end (the rubber tubing and the mouthpiece are supplied along with the calibrated pipettes). 

(vii) Spray the calibrated pipette with 70% (vol/vol) ethanol and wipe off with a Kimwipe. 

(viii) Attach the calibrated pipette to the other end of the mouth pipette. 

(ix) While viewing with the dissecting microscope, break up each colony into tiny fragments with the calibrated pipette and aspirate the fragments by suction. 

(x) Plate the fragments from each colony into a separate well of the 24-well MEF plate prepared in Step 57B(i).<br> <br>CRITICAL STEP Use a separate calibrated pipette for each clone to avoid contamination. 

(xi) After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs. This will identify the colonies that were not picked. 

(xii) If required, carry out further passaging and expansion of the clones for freezing and DNA extraction using 0.1% (wt/vol) Dispase solution, as described in Step 56B(xâxviii), using larger wells and dishes as needed. ESCs/iPSCs should be passaged twice a week to prevent them from differentiating. We typically passage ESCs/iPSCs to a 6-cm dish<br>for DNA extraction.