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Plasmid Extraction with Magnetic Silica Beads - No Phenol Extraction,Reducing Hazardous Waste |
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Description
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.
Features
- Quick, Simple Extraction
MagExtractor® -Plasmid- is based on the principle that magnetic silica beads bind
plasmid DNA, providing simple extraction of plasmid DNA in a short time.
- No Phenol or Chloroform Extraction
This kit does not require the use of harmful phenol or chloroform so there is no
hazardous waste problem.
- Produces High Purity Plasmid DNA
The plasmid DNA extracted with this kit contains hardly any impurities such as genomic DNA, RNA or proteins.
Procedure
This kit, as a manual kit, extracts high purity plasmid DNA according to the following steps.
- Based on a similar principle to the alkali/SDS method, dissolve and neutralize the bacteria.
- Remove the supernatant dissolved in the plasmid DNA by centrifugal separation.
- 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.
- Wash beads with Washing Solution and remove unwanted proteins, salts, etc.
- Elute and recover plasmid DNA from the beads with sterile water or low salt buffer, etc.
One-Point Advice
- B/F Separation
Manual use of this kit is simplified with a magnetic beads separation stand.
B/F separation may be performed using a compact desktop centrifuge instead of a magnetic beads separation stand.
- Electrophoresis of Extracted Plasmid DNA
Since the recovered plasmid DNA solution may contain some ethanol depending on the restriction endonuclease digestion grade method, it is possible that samples may not be immersed in the well with a standard loading dye. The use of the loading dye supplied in this kit is recommended.
- Sample Amount for Extraction
Excessive sample amounts may result in reduced plasmid DNA yields. We recommend a proper amount of sample be used
Applications
- Plasmid DNA Extraction from Bacteria such as E.coli
Recovered plasmid DNA can be used directly in various enzymatic reactions and transformations.
<Example Operations>
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 µg 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.
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