The High-Density Membrane (RPCI-22 Mouse BAC) from Resgen (now part of Invitrogen) was purchased by our lab as an alternative to generating a genomic DNA library ourselves. By screening this membrane with a radiolabeled probe, we were able to quickly and efficiently identify clones of interest. The RPCI-22 membrane is actually an eight-membrane set that represents a mouse BAC library that has been enriched for low abundance genes. These membranes are made by spotting bacteria from 72 plates (382-well) onto a 22 cm x 22 cm Hybond N+ membranes, generating 27,648 unique colonies on one membrane. Each membrane consists of six fields, and each field is made of colony DNA from the clones in twelve 382-well plates. The membranes come with double-stranded positive control DNA (see below) to help orient the membrane.
Screening the membranes require a number of reagents that are not included. Also, the membranes are fairly large, so it is best to use the largest roller bottle possible in the hybridization oven. Probes can be made from a variety of methods such as purified DNA inserts, BAC and PAC insert DNA, alu-PCR products from YACs, or, in our case, oligonucleotides of more than 40 base pairs. Each clone is spotted twice in a unique pattern, allowing for the easy identification of plate addresses and the elimination of false positives. The positive control DNA probes are 500 bp DNA fragment that are spotted on all four edges of the membrane as reference marks for rows and columns. There are 48 reference marks on each side of the membrane as identified by the positive clones. On the right-hand edge of the membrane, in addition to the reference marks, the positive clones are spotted in a unique pattern that can be used as identification marks to help identify which membrane of the eight membrane set it is.
According to the Resgen protocol, one hour of hybridization is good enough to identify clones. However, in our experience, we recommend incubating the probes with the membranes for 16 hours at 42 C to ensure that all the positive clones are the edges of the membrane can be visualized. This is important because the positive clones allow you to accurately label the rows and columns and the identity and orientation of each membrane. Conveniently a plastic alignment sheet is provided to help orient the autoradiogram and identify the plate, row, and column of the positive clones. Once the positive clones are identified, the individual clones can be purchased by Invitrogen.
The technical staff at Resgen has been very helpful with addressing any questions we have. They even allowed us to send the files of our hybridized membranes to them so they could determine if we got any positive clones. In addition, when one of the membranes had some background problems, Resgen sent us a replacement membrane free of charge. The protocol supplied with the membranes is very easy to follow and the whole screening process took only one week. We highly recommend the Resgen High-Density Membranes as an alternative to creating your own genomic DNA libraries (Resgen also has membranes with libraries made from human and rat BAC libraries).
Hee Chul Lee
Graduate Student
Dept. of Biochemistry
NYU School of Medicine