- Item Description
- Replacement Columns and Buffers
- Sample Size: 50 – 150ml of cultured bacterial cells
- Operation: anion-exchange resin column, gravity flow
- Expectant Yield: up to 400µg of pure transfection grade plasmid DNA form 100ml cultured bacterial cells
- Elution Volume: 500µl – 2ml
- Operation Time: 80 minutes or less
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The I-Blue Midi Plasmid Endo-Free Kit (Endotoxin Free) uses pre-packed anion-exchange resin columns to purify plasmid DNA from 50 – 150 ml of cultured bacterial cells. I-Blue Lysis Buffer (an option color indicator) is included with the kit in order to prevent common handling errors, ensuring efficient cell lysis and SDS precipitation. The I-Blue lysis buffer is added druing the re-suspension step, then PM2 buffer is added. After the addition of the PM2 buffer, the sample will begin to turn blue during mixing, when the sample is completely blue complete lysis has been achieved. Colorless areas or brown clumps indicate incomplete lysis and mixing should continue until the sample is entirely blue. During the neutralization step, PM3 buffer is added, and shaking the tube vigorously will begin to turn the sample back to a neutral color. Once the sample is completely colorless, neutralization is complete. PER buffer can now be added to remove endotoxins. Binding, washing and elution of the purified Plasmid DNA can now be done.
Due to the high efficiency of the Midi Column, the entire procedure can be completed in 80 minutes without ultracentrifuges, HPLC, or other toxic reagents. The purified plasmid DNA is suitable for transfection, sequencing reactions, ligation, PCR, in-vitro transcription, microinjection, restriction enzyme digestion, and gene gun.
The quality of the I-Blue Midi Plasmid Endo-Free Kit is tested on a lot-to-lot basis, by isolating plasmid DNA from a 50 ml overnightE. coli(DH5α) culture, containing pBluescript (A600>2U/ml). Following the purification process, a yield of more than 250 µg is obtained and the A260/A280 ratio is between 1.8-2.0. The purified plasmid (1 µg) is used in EcoRI digestion and analyzed by electrophoresis.
|Specifications||I-Blue Midi Plasmid Kit (Endotoxin-Free)|
|Binding Capacity:||500 µg|
|Endotoxin Level:||<0.10 EU/µg|
|Sample Type:||cultured bacterial cells (high-copy = 50-100 ml, low-copy = 100-150 ml)|
|Sample Concentration:||50-150 ml|
|Fragment Size:||1-20 kb|
|Typical Yield:||200-500 µg|
|Elution Volume:||500 µg- 2 ml|
|Operation Time:||<80 minutes|