This kit is for extraction of high purity plasmid DNA from bacteria such as E.coli. The recovered plasmid DNA can be used directly in enzymatic reactions including restriction endonuclease digestion and PCR sequencing and transformation. As this kit employs the principle that magnetic silica beads bind plasmid DNA present in a lysate solution, it is not necessary to perform deproteinization using harmful reagents such as phenol, or ethanol precipitation. Further, high-speed centrifugation is minimized, and if the MFX Series automatic nucleic acids extraction system is used it is not necessary at all. This kit is suitable for the MFX series automatic nucleic acids extraction system and can also be used as a manual kit for B/F (solid-liquid) separation using a magnetic beads separation stand.
1. Restriction Endonuclease Processing of the Extracted Plasmid
Plasmid was extracted from 1.2 ml of the solution cultured overnight of JM109 transformant harboring pUC18, using this kit and another commercially available kit. There was hardly any contamination by proteins, genomic DNA or RNA, and the plasmid DNA yield using this kit was equal to or higher than that obtained with the other commercially available kit.
In addition, part of the obtained plasmid was digested with a restriction endonuclease, and the plasmid extracted with this kit as well as the other commercial available kit were confirmed to be completely digested.
The result shows the use of this kit allows extraction of high purity plasmid from bacteria such as E.coli.
2. Sequencing Analysis of the Extracted Plasmid
An ABI PRISM 310 Genetic Analyzer was used to perform sequencing using 0.5 g of pUC18 extracted with this kit. The sequence primer used was M13 Primer P8 (Code No. PRM-005) and the sequence kit was dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit (PE Applied Biosystems).
The results show that plasmid extracted with this kit is of sufficient quality to be used in auto-sequencing.
This kit, as a manual kit, extracts high purity plasmid DNA according to the following steps.
1. Based on a similar principle to the alkali/SDS method, dissolve and neutralize the bacteria.
2. Remove the supernatant dissolved in the plasmid DNA by centrifugal separation.
3. Add Adsorption Solution containing chaotropic reagents and Magnetic Silica Beads and mix for the plasmid DNA in the sample to adhere to the silica's surface.
4. Wash beads with Washing Solution and remove unwanted proteins, salts, etc.
5. Elute and recover plasmid DNA from the beads with sterile water or low salt buffer, etc.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.