DNA Pool production
Pooling strategy permits to decrease the number of PCR reactions needed to screen an entire genomic library by PCR. Either cDNA or BAC libraries can be pooled in several dimensions: plates, row and columns. After transferring the pools in microplates, the DNA is extracted from the samples or amplified by a “whole genome DNA polymerase”. All the pooling and transferring steps are performed by a pipetting automate and are electronically tracked.
The DNA Pools are checked by concentration measurement and validated with reference primers. Screenings can be performed by traditional or real time PCR. Positive coordinates show significant amplifications and melting curves or gel deposit allow validation of clones containing the sequence of interest.
CNRGV can provide :
- « Ready to use » pools DNA from various genomic libraries: Tomato, Wheat, Sunflower, Sugarcane, Rapeseed, Pepper...
- Pool producing service for your own genomic library
Various pooling strategies can be used, depending on the size of genomic libraries.
2D pools are produced by pooling 384 clones from each microplate from one BAC library alltogether, and then pooling these plate pools according to a 2D matrix. After DNA amplification, PCR screening of the 2D pools allows the identification of some plates containing a specific clone of interest.
For each positive plate, secondary row and column pools are then produced and screened in order to identify a positive clone.
2D pooling is easy to be carried out and is really useful for screening large genomic collection such as wheat for a minimal cost.
3D pools are produced by pooling clones of a BAC library subset according to a 3D matrix in rows, columns and plates. The pools are then transferred in microplates and amplified by a whole genome polymerase. The screening of 3D pools allows direct identification of positive clones.
The number of sets and plates per set depends on the library size and on its coverage.
Please contact us for more details on available pools or on your specific project.