Preparation of DNA
The portion of the construct that comprises the DNA targeting construct (including the 5’ homology region, the selection cassettes, and the 3’ homology Region) need to be removed from the vector sequences by unique restriction enzyme (RE) sites.
The alternative is to linearize the construct at the 5’ end (i.e., the vector sequences are at the 3’ end of the DNA when made linear by RE digestion).
Completely digest 250-500 µg of DNA with (s) the appropriate enzyme and buffer(s).
Separate the DNA targeting construct from the vector sequence by running it overnight on a 1% LMP gel.
Isolate the DNA piece from the LMP gel by Qiagen Extraction Kits or GeneClean.
Important: make sure all the ETOH from washes is removed!
Quantitate the DNA by loading an aliquot on a 1% agarose checking gel with markers of known concentration (e.g., Gene Choice markers from PGC).
Document the DNA concentration with a photo to be submitted with the DNA for electroporation.
The targeting DNA will be mixed with ES cells at three concentrations and electroporated by a standard protocol.
The Facility needs a minimum of 250 µg of isolated targeting DNA per project to obtain approximately 200 selection resistant ES clones (usually G418 resistant) for screening for the correct targeting event.