Required Materials Upon Receiving of Project
(80 micrograms of DNA, concentration > 0.5 mg/ml)
The investigator should provide 80 micrograms of plasmid DNA, preferably prepared with a Qiagen kit.
Picture of the DNA Construct:
The user is required to provide a gel picture of the plasmid digested with a restriction enzyme such as EcoRI or BamHI. This digestion should be done on the DNA prep to be used for electroporation.
Positive Selection Cassette:
The Neomycin resistance gene is the most commonly used positive selection marker but the Hygromycin resistance gene can be an alternative. Recommended promoters include: PGK-1, TK and chicken β-actin.
Physical Map of the DNA:
The user is required to provide a physical map of the plasmid DNA containing the targeting vector. This map should show clearly the selectable marker(s) and their promoters as well as the flanking genomic DNA sequences.
Physical Maps of the Genomic Locus:
A physical map of the wild type locus and a map of the expected targeted allele should also be provided.
DNA Probe for Screening:
(100 ng of probe DNA)
The user must provide at least 100 ng of probe DNA that will be used by the Facility to screen the selected clones. A picture of the Southern Blot of mouse genomic DNA hybridized with the probe should also be provided.
Signed Request Form:
Complete the Request Form and fill in all the appropriate information. The PI must sign this completed Request Form prior to any scheduling of the electroporation.