Molecular Cloning Facility
- Mutagenesis: Introduction of point mutations, insertions (including epitope tags) or deletions in plasmids.
Mutagenesis work order (for alterations up to 7 bases)
Recombinant PCR work order (for alteration >7 bases)
- Subcloning: DNA fragments will be subcloned into the vector of your choice and recombinant clones will be verified by restriction analysis.
- A variety of common vectors are available in the lab, or can be ordered if requested. Starting materials can be plasmid/viral DNA, genomic DNA, or PCR products.
- If appropriate restriction sites are already present, fragments will be cloned directly.
- If requested, restriction sites will be introduced via PCR. Fragments may be subcloned into a TA vector as an intermediate, or as the final product if desired.
- Plasmid preps: Generally we provide 2 clones resulting from mutagenesis or subcloning procedures. Additional clones, or larger scale preps, are available upon request
- Real time qPCR: An ABI 7500 Fast machine is available in the core facility. Genomic DNA, viral nucleic acids and mRNA can be quantitated against an endogenous control (e.g., GAPDH) or standard curve
- Reverse transcription: RNA will be converted to cDNA with reverse transcriptase
- Construction of libraries:
- Genomic DNA Library: Purified genomic DNA of high molecular weight should be supplied by the investigator. The library will be constructed in a lambda or cosmid vector of your choice.
- cDNA Library: Purified total RNA should be provided by the investigator in sufficient quantity. We will isolate the mRNA and construct the cDNA library in lambda vector of your choice and amplify the library.
- Other services: If there are things you would like to try, or would like us to try, feel free to come by to discuss!
Enzymes, Reagents, and Molecular Products:
- Competent E. coli for transformation:
- DH5alpha, XL1-Blue, and BL21(DE3) are available at all times. Other strains can be requested, in batches of ~10 ml.
- We operate an Invitrogen (Gibco/BRL) Supply Center that is stocked with enzymes, reagents, and other molecular products. If it isn't in stock, we can order it.
- Frequently used Qiagen kits (miniprep, PCR cleanup, gel extraction) are available in stock for purchase or can be ordered.
- Molecular weight markers: Two custom-made molecular weight markers are available.
- Workshops: The MCF periodically conducts workshops on various molecular biology related techniques. All interested faculty, staff, and graduate students are welcome to participate in these workshops. A memo will be circulated prior to each workshop with all pertinent information.