ANIMAL GENETICS DEPARTMENT (in french)
INRA BAC-YAC RESOURCE CENTER
LIBRARY RESOURCES :
1) Porcine BAC library
2) Porcine YAC library
3) Bovine BAC library
4) Bovine YAC library
5) Sheep BAC library
6) Rabbit BAC library
7) Goat BAC library
8) Horse BAC library
HOW IS THE SCREENING PERFORMED ?
In each library, clones are ordered in 96-well microplates. Each clone is defined by specific coordinates or address : microplate number and row-column position in the microplate.
Determining a clone's address - screening - is performed by PCR on pools of clones. Indeed, there is a large number of clones in a library, and the number of amplification reactions must be minimized. So, clones are pooled by groups of 20 to 24 96-well microplates corresponding to Superpools. Each Superpool is subdivided in 8 row pools, 12 column pools and x plate pools, x ranging from 20 to 24.
Library screening and clone isolation are performed in 4 steps :
1. PCR on genomic DNA to check conditions
2. PCR on the Superpools
3. PCR on the 8 row pools, the 12 column pools and the x plate pools corresponding to the positive Superpool. Combination of these 3 informations gives the clone's address.
4. The positive clone is then streaked on an LB-agar-chloramphenicol plate and two individual colonies are again tested by PCR. One colony is chosen and sent on an LB-agar-chloramphenicol stab.
Request for using the libraries
Request for using the libraries should be addressed to : Marco MOROLDO (firstname.lastname@example.org).
Our requests for the screening are the following :
1) please indicate :
Your full name and address (to send the clones).
Reference of the primers.
Marker or gene name : we just want to know if it is not a marker or a gene that we are currently working on or we have already pulled out from the library. If it is the case, it could save time.
Which library should be screened.
A rapid description of your project and particularly if you need only one clone by primer pair (localization studies) or all the clones we can identify for one primer pair (contig studies).
Size of amplified fragment.
PCR conditions for all primer pairs.
Possibly, the amplification result on genomic DNA.
2) send approximately 100 Ál of each primer (200 Ál by primer pair) at 100 ÁM (100 pmoles/Ál). It may represent a large amount but it allows us to work in safer conditions. We prefer to avoid to ask you to send more primers in case of amplification problems. If the primer concentration you have is lower, please adjust the volume to be sure to send the same amount. They can be sent in an aqueous solution in tightly closed tubes (covered by parafilm). Fluorescent labeled primers can be used.
SHIPMENT - PAYMENT (for laboratories outside Europe only)
Concerning payment, you will only be billed for clone shipping charges. If you have a FedEx or DHL account number, please let us know. You will be directly charged by these companies. Delay from France is approximately 2 or 3 days.
Since the library screening is free of charge and the clones are available for the scientific community, reference of the library has to be mentioned in the publication as well as the origin of the BAC clones in the acknowledgments section i.e. : the BAC-YAC Resource Center of the Animal Genetics Department of the INRA. The person who performed the screening can also be mentioned in the acknowledgment section or be part of the authors. It depends of course of the importance of the BAC(s) clone(s) recovery in the whole work.
Information concerning the BAC vector used : pBeloBAC11
Construction and characterization of a human bacterial artificial chromosome library.
Kim UJ, Birren BW, Slepak T, Mancino V, Boysen C, Kang HL, Simon MI, Shizuya H
Genomics 1996 Jun 1;34(2):213-8
Restriction site : HindIII
BAC DNA extraction protocols