FAQ AquaPlasmid, AquaGenomic, & AquaRNA are innovative aqueous solutions for nucleic acid isolation and purification.

AquaPlasmid, AquaGenomic, & AquaRNA aqueous solutions for nucleic acid isolation and purification

- Frequently Asked Questions -

The following are some commonly asked questions about AquaPlasmid^TM, AquaGenomic^TM, AquaRNA^TM, and AquaBluer^TM. Please read through these questions carefully. The answers provide additional helpful tips and useful information for the successful use of AquaPlasmid^TM, AquaGenomic^TM, AquaRNA^TM, and AquaBluer^TM.

AquaPlasmid FAQ

1. Do I need to store the AquaPlasmid kit at 4 or -20° C?

Do not store the AquaPlasmid kit in a refrigerator or freezer. All solutions in the kit are stable at room temperature (~22° C). The reagent bottles should be tightly capped. If precipitates appear in AquaLysis solution due to cold weather, incubating at 37° C for 5-10 minutes will clear the solution.

2. Do I need to keep all the solutions and samples on ice while carrying out the isolation steps?

No, all the samples and solutions, including isopropanol and ethanol, should be kept at room temperature. All the procedures should be performed at room temperature.

3. Does AquaLysis contain sodium hydroxide?

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.aquaplasmid.com.

4. Does AquaPlasmid contain RNase A?

No. 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. Will vortexing the cell lysate and cell debris shear genomic DNA and cause genomic DNA contamination?

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. Is it critical to follow the specified incubation and centrifugation times exactly?

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. How should I scale up AquaPlasmid and companion solutions?

You may use AquaPlasmid miniprep protocol as a reference. For each milliliter of culture, you use 50 ul 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. Should I use centrifugation to rinse the DNA pellet? I am afraid that my pellet may lose during ethanol rinse.

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 3 times. For midi and maxiprep ensure that the DNA pellet remains attached at the bottom of the tube before discarding the ethanol solution. For miniprep, the DNA pellet may be invisible.

9. How pure is AquaPlasmid isolated plasmid DNA?

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 2.0.

10. Is AquaPlasmid isolated plasmid DNA free of endotoxins?

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. Can I estimate DNA yield using a UV spectrophotometer?

Plasmid DNA yield often is over-estimated from its OD values at 260 nm due to the contamination 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 ultra pure and can be accurately measured with a UV spectrophotometer.

12. What plasmid DNA yield can I expect using AquaPlasmid?

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. What QC tests do you use to certify your products?

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 2.0 and be readily digested with restriction enzymes.

AquaGenomic FAQ

1. Do I need to store the AquaGenomic kit in the refrigerator or freezer?

No, AquaGenomic Solution is stable at room temperature (~22° C). If precipitates appear due to cold weather, incubating at 37° C for 5-10 minutes will clear the solution.

2. Should I keep the solutions and samples on ice while carrying out the isolation?

No. Most of the isolation steps should be performed at room temperature (~22° C). For DNA isolation from animal cells, cell lysis at 60° C may increase the yield by ~25%. For DNA isolation from cells with a cell wall, such as bacterial and plant cells, the cell lysis should be carried out at 60-90° C for 15-30 minutes.

3. Does AquaGenomic Solution contain Proteinase K?

No. AquaGenomic Solutions can be used to extract DNA from cells and tissues without needing protease digestion. This reduces the time required for genomic DNA isolation from solid tissues from a full day to only ~15 minutes.

4. What type of homogenizer do you recommend for using with AquaGenomic?

Pestle-and-tube homogenizers produce more homogeneous samples. However, if you do not have a homogenizer handy, you can smash the tissue with a pair of tweezers. Another option is to use the 60° C extraction method, which does not require a homogenizer but a few hours of 60° C incubation. At the end of incubation, you may add some glass or ceramic beads to the sample and vortex vigorously to disrupt the tissue.

5. Does AquaGenomic Solution contain RNase A?

No. AquaGenomic Solution can remove most RNA contaminant. Trace amount of RNA would not interfere with most genomic DNA applications.

6. Why do I need to purchase a separate reagent for fecal DNA isolation?

Due to the presence of large amount of enzyme inhibitors in feces and soil, DNA isolation from these samples are particularly challenging. Precipitation of the DNA with isopropanol cannot remove some of the inhibitors and a specially formulated AquaPrecipi Solution (Product # 3015) is required for selective precipitation of the DNA to remove these enzyme inhibitors.

7. Can I use water for RBC lysis?

For fresh blood: YES. Simply add 10 volumes of deionized water to the blood sample, vortex 20-30 seconds, centrifuge to pellet the cells, remove the red supernatant, and then proceed to extract DNA using AquaGenomic. For frozen blood: NO. Thawed RBC will not be lysed with water and you need to use our RBC Lysis Solution (Product # 4015), which is a modified hypotonic solution that lyses either fresh or frozen RBC.

8. I am concerned about cross-contamination using homogenizers, any tip?

Yes. Between uses, you should thoroughly wash the homogenizer with soap and running water, soak it in 10% bleach for ~5 minutes, and then rinse it with running deionized water. This will prevent cross-contamination of the genomic DNA. If you still feel uneasy, you can use the 60° C extraction and bead milling method to disrupt the tissues.

9. My final DNA pellet does not dissolve well in water, what can I do?

You may soak the final DNA pellet in water or TE buffer at room temperature for 1-16 hours, pipette up-and-down or vortex vigorously to fully disperse the pellet, and then centrifuge at 12000xg for 2 minutes to pellet any insoluble.

10. What genomic DNA yield can I expect using AquaGenomic?

Approximately 10 ug DNA can be obtained from 1-2 million cultured cells, 300-400 ul of whole blood, 1 ml of overnight microbial culture, 5-10 mg of animal tissues, or 10-20 mg of plant tissues. DNA yield is dependent on the number of nucleated cells in the sample and may vary at different cell cycles and for different cell types.

11. How pure is the genomic DNA isolated by AquaGenomic?

Typical A260/A280 of AquaGenomic purified DNA is 1.6-1.8. The isolated genomic DNA is free from most cellular contaminants and enzyme inhibitors. However, there may be some small RNA contamination, if RNase A treatment is not included.

12. What QC tests do you use to certify your products?

Each lot is tested to ensure its performance and reliability in isolating genomic DNA. The isolated DNA (a) should have an A260/A280 ³ 1.6, and (b) should be readily digested with restriction enzymes.

AquaRNA FAQ

1. Should I keep the AquaRNA kit in the freezer?

No, AquaRNA Solution is stable at 4° or 22° C.

2. How does AquaRNA work?

AquaRNA is a multifunctional aqueous solution. It lyses the cells, inactivates the RNases, and extracts DNA, RNA, and proteins in one step. Total DNA/RNA are precipitated from the clear lysate with isopropanol and proteins in the supernatant are subsequently precipitated with acetone.

3. Do I need to add RNase Inhibitor to the final RNA solution?

No. AquaRNA is very effective in inactivating and removing RNases. You will discover that your RNA is no longer problematic and it is no longer tricky to work with RNA. You could even leave your RNA samples at room temperatures for days without noticing any RNA degradation.

4. How should I remove DNA from the RNA preparation?

If DNA removal is desired, you may use RNase-free DNase I to degrade the genomic DNA in the RNA preparation. However, DNA removal is unnecessary with the use of a simple and elegant 5' tailed RT-PCR [Knuchel and Ansari. Tailed RT-PCR for the quantitation of chloramphenicol acetyl transferase (CAT) mRNA, In "Methods in Molecular Medicine, Vol.XX - Quantitative PCR Protocols," Humana Press, (Chapter 18):1-11, 1997. Hurteau and Spivack. mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts. Anal Biochem. 2002 Aug 15;307(2):304-15. Chen, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research 2005 33(20):e179 (FREE Full Text and Supplementary Data for primer design)].

5. Can I use AquaRNA with my Trizol® (Invitrogen) to inactivate RNases.

Yes, you could use AquaRNA in conjunction with other RNA isolation methods during extraction (1 vol. of AquaRNA and 1 vol. of phenol or Trizol or other lysis solution) to irreversibly inactivate RNases and you should at least double the RNA yield.

6. What QC tests do you use to certify your products?

Each lot is tested to ensure its performance and reliability in isolating DNA/RNA. An RNase assay is performed (incubating RNA prep in 1x DNase I buffer at 37° C for 15 minutes and observing RNA integrity in 0.7% native agarose gel by electrophoresis) to ensure no RNase contamination.

AquaBluer FAQ

1. Should I keep AquaBluer™ in the freezer?

AquaBluer™ solution is stable at 4-22 °C for 12 months. However, if stored at –20 °C, its shelf life could extend indefinitely. It is important to keep the solution in complete darkness and minimize its light exposure.

2. How much cells should I seed in each well?

It depends on how fast the cells propagate and how long you need to treat the cells with a test compound. We find that by adjusting the seeding cell density to produce ~1000 RFU values in untreated control at the end of 4-hour incubation with AquaBluer™ is optimal.

3. Do the plates need to be equilibrated at room temperatures before reading?

Plate temperature could affect the fluorescence reading. Therefore, you need to be consistent with all plates – either cool them all down before reading or don’t.

4. Can AquaBluer™ treated culture be used for other assays?

AquaBluer™ is nontoxic to the cells and you should be able to use those cells for subsequent cell analysis. It is unlikely to interfere with other bioassays, but you need to test it for your particular assays to be sure. We routinely recover the media containing AquaBluer™ for multiplex cytokine ELISA assays.

5. I don’t have the exact 540ex/590em fluorescence filter set, what can I do?

You may use any fluorescence filter set covering 550±20nm for excitation and 600±20nm for emission. Similarly, for absorbance reading, you could use a 570±20nm and 600±20nm filter set.

6. Can I use AquaBluer™ to assess the viability of other organisms?

Yes, AquaBluer™ may be used to assess the viability of bacteria, mycobacteria, fungi, animal and plant cells, and even tissues and organs.

7. What QC tests do you use to certify your products?

Each new lot of AquaBluer™ has an A600(oxidized)/A570(reduced) ratio >1.3.