What are the recommended culture and buffer volumes for a very low-copy plasmid?
Very low-copy plasmids and cosmids of less than 10 copies per cell often require large culture volumes to yield significant amounts of DNA. The recommended conditions below are suitable for QIAGEN-tip 100 or QIAGEN-tip 500, and use centrifugation to clear lysates rather than QIAfilter Cartridges, due to the large culture volumes. After alkaline lysis, there is an additional isopropanol precipitation step to decrease the amount of lysate before DNA is bound to the QIAGEN-tip. Please follow the protocol for 'Very Low-Copy Plasmid/Cosmid Purification Using QIAGEN-tip 100 or QIAGEN-tip 500' in the QIAGEN Plasmid Purification Handbook. Culture volumes and tip sizes are selected to match the quantity of expected DNA with the capacity of the QIAGEN-tip.
Parameters for purification of very low-copy plasmids and cosmids of less than 10 copies per cell
|Required DNA yield*||Up to 100 ug||Up to 500 ug|
|500 ml||2.5 liters|
|Buffer P1||20 ml||125 ml|
|Buffer P2||20 ml||125 ml|
|Buffer P3||20 ml||125 ml|
|Buffer QBT (for equilibrating)||4 ml||10 ml|
|Buffer QC (for washing)||2x 10 ml||2x 30 ml|
|Buffer QF (for elution)||5 ml||15 ml|
* For very low-copy plasmids, expected yields are 20–100 µg for the QIAGEN-tip 100, and 100–500 µg for the QIAGEN-tip 500.
† Volumes of lysis Buffers P1, P2, and P3 are higher than in the standard protocols in order to efficiently lyse the large number of cells required for purification of very low-copy plasmids and cosmids.