Colony screening

Hybridoma SN should preferably be used for colony screening because there is always the possibility of false-positive colonies being selected using ascites. Ascites contain a lot of antibodies with unknown specificity, which could bind phage from the library. 

Cell lines that are carried forward as potential production lines are sub-cloned. Cells are plated at an average of 0.3 cells per well in 96-weU plates, and out-growing colonies screened, expanded, frozen, and tested as described for the initial clones. 

Fig. 1. Colony screening. E. colt, colonies transformed with a plasmid carrying a portion of the proto-oncogene Nras hybridized with a l.S-kbp HRP-labeled Nras probe as described in the text. 1-min exposure on Hyperfilm-MP.

Fig. 1. Colony screening. Top three filters E. coli colonies transformed with pSP65 containing a portion of the human Nras proto-oncogene lifted onto Hybond-N. Bottom two filters E. coli colonies transformed with pSP65 lifted onto Hybond-N. Both sets were hybridized with 10 ng/mL of an Nras specific 2S-base oligonucleotide labeled at the 3 end with fluorescein-dUTP. Hybridized for 2 h at 42°C stringent wash IX SSC, 0.1% SDS at 42"C 10-min exposure on Hyperfilm-MP.

Individual colonies screened for required gene or its protein product... 

Perform colony screening PCR on at least 24 colonies nsing the start and stop primers for one of the BBs. 

Isolate plasmid from the positive clones of colony screening PCR and analyze the recombinant plasmid by restriction enzyme digestion for the presence of correct-sized insert. 

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