products | nucleic acid and protein tools | Nucleic Acid Purification | AquaPlasmid™

Nucleic Acid Purification

AquaPlasmid™
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A novel aqueous solution-based tenology for isolation and purification of ultrapure plasmid DNA

Overview

AquaPlasmid FAQ

1. Q.: Do I need to store the AquaPlasmid kit at 4° or -20° C?
   A.: Do not store the AquaPlasmid™ kit in a refrigerator or freezer. All solutions of the kit are stable at room temperature (~22° C). The reagent bottles should be tightly capped.
   
   
2. Q.: Do I need to keep all the solutions and samples on ice while carrying out the isolation steps?
   A.: No, all the samples and solutions, including ethanol, should be kept at room temperature. All the procedures should be performed at room temperature.
   
   
3. Q.: Does AquaLysis contain sodium hydroxide?
   A.: Yes, AquaLysis is a modified alkaline lysis solution, which contains 1% of sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS). You shall handle AquaLysis Solution as other chemicals using Good Laboratory Practice (GLP). Always wear rubber gloves and laboratory coat when handling AquaLysis and AquaPlasmid solutions. MSDS for AquaLysis and AquaPlasmid Solutions are available at our website www.mobitec.com.
   
   
4. Q.: Does AquaPlasmid contain RNase A?
   A.: RNase A contamination of laboratory equipment and work area is a major concern for any RNA work. You can be sure that neither AquaLysis Solution nor AquaPlasmid Solution contains RNase A.
   
   
5. Q.: Will vortexing the cell lysate and cell debris shear genomic DNA and cause genomic DNA contamination?
   A.: AquaPlasmid Solution is highly effective in removing genomic DNA contamination. You can easily verify it by using AquaPlasmid kit on E. coli cells that do not harbor plasmids. Vortexing or shaking the lysate and debris ensures cell lysis and plasmid DNA recovery.
   
   
6. Q.: Is it critical to follow the specified incubation and centrifugation times exactly?
   A.: The incubation and centrifugation times given in the protocols are the minimum. You may use longer incubation and centrifugation times. However, do not incubate cell lysis more than 10 minutes.
   
   
7. Q.: How should I scale up AquaPlasmid and Aqualysis Solutions?
   A.: You may use AquaPlasmid miniprep protocol as a reference. For each milliliter of culture, you use 50 ml of deionized water to resuspend the cells, 30 ul of AquaLysis Solution to lyse the cells, 30 ul of AquaPlasmid Solution to extract plasmid DNA and precipitate cell debris, and then 30 ul of AquaPlasmid Solution to precipitate plasmid DNA from 100 ul of cleared cell lysate.
   
   
8. Q.: Should I use centrifugation to rinse the DNA pellet? I am afraid that my pellet may lose during ethanol rinse.
   A.: It is unnecessary. Gently shoot the ethanol solution into the tube along the sidewall, rotate it, and then flip the tube to discard the ethanol. Repeat the ethanol rinse 2-3 times. However, for midi and maxiprep ensure that the pellet remains attached at the bottom of the tube before discarding the ethanol solution.
   
   
9. Q.: How pure is AquaPlasmid isolated plasmid DNA?
   A.: The plasmid DNA is essentially free of cellular impurities and other enzyme inhibitors. Plasmid DNA isolated by AquaPlasmid™ usually has an A260/A280 ratio of 1.9 - 2.0.
   
   
10. Q.: Is AquaPlasmid™ isolated plasmid DNA free of endotoxins?
    A.: AquaPlasmid does not rely on charge interaction to selectively bind plasmid DNA and co-purify negatively charged endotoxins. AquaPlasmid isolated DNA contains ≤0.1 EU endotoxins per microgram of DNA, which is considered endotoxin free.
    
    
11. Q.: Can I estimate DNA yield using a UV spectrophotometer?
    A.: Plasmid DNA yield often is over-estimated from its OD values at 260 nm due to the contaminations of protein, RNA, and RNase A generated nucleotides. Unlike DNA isolated with other kits where RNase A is used to degrade cellular RNA, AquaPlasmid isolated DNA is ultrapure and can be accurately measured with a UV spectrometer.
    
    
12. Q.: What plasmid DNA yield can I expect using AquaPlasmid?
    A.: For high copy number plasmids, typical plasmid DNA yield is ~10-20 ug DNA per ml of overnight Terrific Broth (TB) culture and ~5-10 ug DNA per ml of overnight Luria-Bertani (LB) culture.
    
    
13. Q.: What QC tests do you use to certify your products?
    A.: Each lot of AquaLysis and AquaPlasmid is tested to ensure its performance and reliability in isolating plasmid DNA. The isolated DNA should have an A260/A280 ratio around 1.9 - 2.0 and be readily digested with restriction enzymes.

See also chapter 6 of our online catalog.

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