Our Approach to Library Clone Pooling Strategy
At Rx Biosciences, our library clone pooling strategy ensures unbiased and accurate PCR-based screening. We start by growing individual clones separately. This step eliminates bias that may result from different growth rates among clones.
After growing the clones, we mix them to create pools and super-pools. This structured library clone pooling strategy helps maintain consistency and simplifies downstream screening steps.
We then isolate DNA from super-pools, plate-pools, and both row and column pools. This process supports efficient identification and tracking of positive clones during PCR screening.
Our method is designed to save time and reduce errors. By using a defined pooling hierarchy, we streamline the clone screening process while ensuring reliable results.
The diagram or explanation of our pooling layout is provided below. This helps visualize the entire strategy and makes implementation straightforward for your research team.
- Eight consecutive 384-wel plates, containing individually picked clones, are pooled together to form a Super-pool plate.
- For each super-pool plate, you will be provided with the corresponding plate pools (8), column pools (24) & row pools (16).
- Plate pool contains the DNA prepared from all the clones present in one of the eight 384-well plates (i.e. eight plate pool DNA for each super-pool)
- Column pool plate contains all BAC clone DNA from the same column of the eight 384-well plates (i.e. 24 column pools for each super-pool)
- Row pool plate contains BAC clone DNA from the same row of the eight 384-well plate (i.e. 16 row pools for each super-pool)
- Both, the glycerol stock and the DNA for the super-pools, plate pools and row &column pools are delivered.
Example: A library of 80 384-well plates will generate:
- 80 Plate pools [80 DNA preps]
- 10 Super-pool plate pools [10 DNA preps]
- 160 Row pools [160 DNA preps]
- 240 Column Pools [240 DNA preps]
Screening Strategy
- First PCR: Set up PCR using super-pool plate DNAs (e.g., 10 reactions to cover 80 plates)
- Second PCR: Once a super-pool is identified, set up PCR using the DNA from plate pools corresponding to the particular super-pool (8 reactions).
- Third set of PCR: Once a plate is identified, use DNA from row and column pools and locate the address of the clone. This will identify the position of the clone in the plate such as if row A and column 8 are positive, then the positive clone is A8.
- Grow positive clones, prepare DNA and confirm by additional methods such as Southern blots or PCR followed by sequencing of the PCR product.
Request a Quote
Ready to start your project? Send us a message and we’ll provide you with a customized quotation for your project’s requirements.