The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high purity plasmid DNA. The purified plasmid DNA can be used for immediate use in all molecular biology procedures such as digestion with restriction enzymes, cloning, PCR, transfection, in vitro translation, blotting and sequencing.
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.
Format | Manual; Solution Based (Scalable) |
Preparation time | 40 minutes |
Equipment required | Microcentrifuge |
Minimum elution buffer volume | 50 µl |
Culture volume for high-copy plasmids | 1–5 ml |
Culture volume for low-copy plasmids/cosmids | 1–10 ml |
Yield is variable and depends both on the particular E. coli strain used and the cell density of the bacterial culture.
Contents |
Storage Temperature (upon receipt)
|
Buffer P1 |
15-25°C |
Buffer P2 |
15-25°C |
Buffer P3 |
15-25°C |
All solutions should be kept tightly sealed and stored at room temperature. These reagents should remain stable for at least 1 year in their unopened containers.
For Research Use Only. Not for use in diagnostic procedures.
Items | Cat. # | Size |
---|---|---|
Plasmid DNA Purification kit (Solution Based) | SCMR002 | 50 Prep. |
Plasmid DNA Purification kit (Solution Based) | SCMR002A | 100 Prep. |
Items | Cat. # | Size |
---|---|---|
Plasmid DNA Purification kit (Solution Based) | SCMR002 | 50 Prep. |
Plasmid DNA Purification kit (Solution Based) | SCMR002A | 100 Prep. |
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