Journal
NATURE PROTOCOLS
Volume 2, Issue 11, Pages 2865-2874Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.409
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Funding
- NCI NIH HHS [T32CA67751, CA109274] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [T32CA067751, R55CA109274, R01CA109274] Funding Source: NIH RePORTER
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Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02-0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4-8 weeks depending on the proliferative capacity of the cell line.
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