Preparation of Plasmids
Our philosophy is that plasmid preps can be performed more efficiently, consistently, and cost effectively by a central facility. Because these are done in the core on a daily basis, we have experience hard to march in individual labs. All the critical steps in the procedure—the little tricks, the danger points—have been worked out, and this results in the highest possible yields from even low copy number plasmids.
Please note: In order to be equitable in providing access to services, we will receive no more than four plasmids at a time from any lab. Any submissions greater than four will be held in reserve and completed at the discretion of the lab director. Thank you.
Our standard method is an RNAse-free alkaline lysis/double cesium gradient protocol. Cesium gradients are less expensive than ion exchange column methods and often give markedly higher yields, particularly with high copy number plasmids that exceed the binding capacity of the gel. The second gradient results in a cleaner product with much higher transfection efficiency, but it can be waived if this level of purity is not required.
The core also has established a protocol for recovery of intact plasmid DNA from large (>40 KB) constructs such as adenovirus. Plasmids are delivered resuspended in sterile TE buffer, with documentation of OD 260/280 and DNA concentration.
Plasmids are ordered by delivering a plate to the core lab. Plates must be clearly labeled with the name of the construct, the antibiotic required, the PI and cost center to be charged for the procedure, and the name of the person to call when the plasmid is ready. Once a plasmid is received, a frozen glycerol stock is prepared, so subsequent requests for that plasmid can be made by phone or e-mail. Turnaround time for orders is about two weeks.
In addition, the core has assembled a collection of frozen glycerol stocks of strains containing frequently used plasmids, some of which are standard vectors; some are for generation of fusion proteins, and some are for reporter genes. These plasmids may be ordered from the core by phone at the same cost as a standard plasmid prep.