BAC Libraries
Why BAC clones are essential
- BAC clones are from one specific allele (100 Kb to 200 Kb).
- Sequencing BAC clones can simplify a complex genome to “BAC size” regions of interest!
- BAC’s allow you to determine haplotype and phase of each chromosome/allele.
Allele-specific assemblies and Chromosome Phasing (linking many SNP’s together onto the proper chromosome) are critical for an accurate genome assembly. Bioinformatic leaders strongly support using allele-specific technologies for all genome assemblies (human, personalized medicine, plants, and animals). Performing NGS on BAC clones enables allele-specific sequencing and accurate assemblies.
We can help you determine a cost-effective research strategy, based on your specific goals. Contact us for more information or to get a quote.
Amplicon Express has made custom BAC libraries from the following organisms (and more!)
Insects
Heliconius melpomene, Papilio glaucus, Heliconius numata, Heliconius cydon, Megaselia abdita, Papilio dardanus, Hypolimnas bolina, Limenitis arthemis astyanax, hessian fly, Drosophila, four different strains of mosquito, six varieties of parasitoid wasps, butterflies and four tick species.
Higher plants
Seven wheat BAC libraries (various strains), several barley strains, nine soybean strains, three maize varieties, Panicum maximum, Helianthus annuus, Capsella rubella, canola strains, ryegrass, banana, sugarbeet, mustard, hops, various grapes, cotton, petunia, various species of Arabidopsis, apple, cottonwood, blueberry, citrus, and many rice strains.
Mammals
+50 human cancer tumor/cell line samples, dozens of knock-out/in mouse strains, buffalo, deer, elk, cat, various rat strains and monotremes like duck-billed platypus and opossum.
Metagenomic, Prokaryotic and Fungal organisms
Chlamydomonas reinhardtii, Staphylococcus aureus, Sodalis glossinidius, Streptomyces fradiae, Sorangium cellulosum, Haemophilus influenzae, a wide variety of Actinomycetes and Streptomyces strains, Anaplasma marginale, bacterial mats from Yellowstone National Park hotsprings, and a series of Soil samples above and below industrial factories on contaminated rivers.
Marine/Freshwater Organisms
4 different marine sponges, Platyhelminthes, annelid worms, barnacles, oyster, jellyfish, mussel, sea anemone, three-spined stickleback, 2 strains of ocean rainbow trout (steelhead), Oikopleura, cod fish, zebrafish, sea bream and hagfish.
Amplicon Express BAC Library Construction: Process and Example Data
Construction of an Amplicon Express BAC library begins with the preparation of High Molecular Weight (HMW) DNA embedded in agar plugs, to get megabase-sized genomic DNA.
We then perform a partial endonucelase enzyme digestion titration series and gel purify a series of ‘zones’ containing digested DNA.
After the ideal conditions for digestion and zone purification are determined, we perform a series of ligation and transformation experiments. We verify the experiments by selecting 28 random BAC clones, preparing BAC DNA, digesting with NotI (a 8-base GC rich cutter that has sites flanking the MCS-Multiple Cloning Site-where the HindIII digested fragments are ligated) and run the BAC DNA on Pulsed Field Gel.
After all this is verified, we perform mass transformations, plating and pick. We use the following robotic platforms: G3 (Genomic Solutions), QPIX2 (Genetix) and MegaPIX (Genetix). The robots do blue/white selection and pick white clones into 384-well plates of glycerol storage solution. The plates of clones are grown overnight (~18 hours), and then we determine the exact plate and well position for all wells that did not grow (we call these ‘missed wells’).
We re-grid a subset of the clones onto 22cm x 22cm agar plates with XGAL+IPTG+Antibiotics and allow the colonies to grow 18 hours, then ‘incubate’ for several days at a specific temp to allow any ‘blue clones’ to be very obvious. We then count the blue clones and enter into a database. Example of Custom Mouse BAC Library QC Data There are several sheets in this file, including one that tracks if any “pins” on the robot are going bad. Your team will get a similar Excel file when the library is completed. The combined missed wells and blue clones give us the total error in the library. The total error must be less than 3% or it is rejected by our Quality Control processes.
Screen the BAC Library by PCR with Pools & Superpools (download the user manual here).
Use BAC clones to get the right assembly!
Amplicon Express’ team of dedicated scientists have made over 2,650 custom BAC libraries and processed more than 57 million high-quality, large insert genomic DNA clones since 1996. We are committed to providing the highest quality BAC library resource on the market in a timely and cost-effective manner.
Our extensive experience extracting and cloning HMW gDNA fragments from thousands of different organisms, has enabled us to optimize DNA extraction, purification and ligation protocols to be successful where others have failed. We love working with marine organisms (fish, invertebrates, sponges, etc), plants with strong secondary/defense compounds, troublesome insects and other difficult critters!
BAC Libraries are an ideal tool for Metagenomic Libraries (each +100Kb insert is from a different organism).
Amplicon Express Custom BAC libraries have average insert sizes between 115 Kb and 180 Kb and are used by thousands of researchers in more than 65 countries around the world. New pop-up window View examples of some of our BAC libraries
We pride ourselves on delivering individualized service and project design from the beginning of the quoting process to our one-on-one technical support years after the library has reached its destination. New pop-up window View our BAC Library Construction Process and Example Data
Screen the BAC Library by PCR with Pools & Superpools (download the user manual here).
Contact us to get a BAC library quote.