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

=== Picking drug-resistant clones  ===

57. Follow option A to pick somatic cell clones; the protocol works efficiently for primary or transformed cell lines. MSCs have been used here as an example. Follow option B to pick pluripotent cell clones. 

(A) Picking drug-resistant clones: somatic cell clones â TIMING 0.5 h<br> (i) Identify drug-resistant colonies by scanning the dishes (from Step 56A(xi)) with an inverted microscope and circle them using a permanent ink pen on the bottom of the dish. 

(ii) Prepare a 24-well plate with 0.5 ml of MSC medium per well planning on one well per colony to be picked. 

(iii) Aspirate medium from the dish containing drug-resistant colonies and wash once with 1Ã PBS. 

(iv) With forceps, place cloning cylinders (see EQUIPMENT SETUP) over all the foci to be picked. Ensure that the cloning cylinders are firmly attached to the dish and that they encircle the colonies. 

(v) Pipette 20 ÎŒl of trypsin-EDTA solution into the cloning ring and incubate for 2 min at 37 °C. Add 30 ÎŒl of MSC medium to neutralize the trypsin-EDTA. 

(vi) Collect the detached cells from the cloning cylinder with a pipette and plate each colony into one well of the 24-well plate prepared in Step 57A(ii). 

(vii) After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs. 

(viii) If required, further culture and passage the clones to bigger dishes for freezing and harvesting DNA. We generally transfer somatic cells to 10-cm dishes for DNA extraction.