The following are some commonly asked questions about our products. Please read through these questions carefully. The answers provide additional helpful tips and useful information for the successful use of these products.

• AquaPlasmid FAQ
• AquaGenomic FAQ
• AquaRNA FAQ
• AquaBluer FAQ
• AquaStool FAQ
• AquaPreserve FAQ

AquaPlasmid FAQ

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

No, AquaPlasmid solution is stable at room temperature (~22 °C) for >1 year. However, if the room temperature is below 18 °C, some precipitation may occur in the AquaLysis solution, which can be re-solubilized by incubating at 37 °C for a few minutes.

2. How should I scale up and down the reagents for other starting culture volumes?

The recommended scaling base is “1 ml culture – 40 ul water – 50 ul AquaLysis – 25 ul AquaPlasmid.” For example, you may pellet the bacteria in 4 ml overnight culture, use 160 ul of water to suspend the bacterial pellet, add 200 ul of AquaLysis to lyse the cells, and add 100 ul of AquaPlasmid to neutralize the crude lysate.

3. Does AquaPlasmid contain RNase A?

No, AquaPlasmid solution does not contain RNase A. You may use AquaPlasmid purified plasmid DNA directly for in vitro transcription.

4. Can I use a low-speed centrifuge to do the minipreps?

Yes, you don’t need a high-speed centrifuge to use the AquaPlasmid methods. You may even use a personal picofuge (1-6 minipreps) or a standard speed-vac (96-well plate HTP minipreps) for all the centrifugation steps.

5. Should I use centrifugation to rinse the DNA pellet?

No, it is not necessary. Gently shoot 70% ethanol solution into the tube along the sidewall away from the DNA pellet to fill up the tube, and then flip the tube to discard the ethanol solution. Repeat the ethanol rinse one time. For midi or maxiprep, make sure that the DNA pellet remains attached at the bottom of the tube before decanting to discard the ethanol solution.

6. How pure is the 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 1.8-2.0 and A260/A230 ratio of 2.0-2.2.

1. Do I need to keep AquaGenomic in the freezer?

No, AquaGenomic Solution is stable at room temperature (~22° C) for 12 months.

2. Does AquaGenomic Solution contain Proteinase K?

No. AquaGenomic can be used to extract DNA from cells and tissues without needing protease digestion. However, if mitochondrial DNA recovery is desired, you will need to add Proteinase K to AquaGenomic solution to 100 ug/ml and incubate the sample at 60-65° C for 1-2 hours during cell lysis and then at 95 for 10 minutes to inactivate the Proteinase K.

3. I am concerned about cross-contamination using homogenizers, any tips?

Between uses, you should wash the homogenizer with soap and running water, soak it in 10% bleach for ~5 min, 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 Proteinase K digestion to disrupt the tissues without using a homogenizer.

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

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

5. Why are some of my DNA preps not amplified?

The most common cause is that the water-insoluble material, which contains PCR inhibitors, in the final DNA solution has not been fully removed. You should centrifuge the DNA solution again before transferring it to the PCR reaction.

6. Can I use AquaGenomic to extract RNA?

1. How should I store the AquaRNA solution?

It may be stored at 4 °C for 12 months. Invert to mix the reagent well before dispensing.

2. Do I need to use ProMelt and ProSink?

ProMelt (Item # 1115) is an ancillary reagent for solubilizing protein pellet precipitated by acetone. It is not required, if you don’t plan to recover the proteins. ProSink (Item # 9030) is a protein precipitating solution for DNA/RNA extraction from blood or other nuclease-rich animal tissues. ProSink is optional, if you extract DNA and RNA from bacteria, cultured cells, or plant tissues.

3. I did not see the 28S and 18S rRNA bands in the gel, why?

The 28S and 18S rRNA bands may migrate with the genomic DNA in a native 0.8% agarose gel. However, if you add some salts (e.g., 30 mM NaOAc, pH unadjusted) to the loading dye, you should get a good separation of the 5S, 18S, 28S rRNA, and the genomic DNA bands. Alternatively, you can do a DNase I digestion to remove the DNA before running the gel.

4. My RNA was degraded, where was the RNase coming from?

To troubleshoot RNase contamination, you may set up a DNase I digestion in 1x DNase buffer. Before adding DNase I, divide the sample into two aliquots and add DNase I to one of them. If RNA degradation is seen only in the DNase I treated sample, your DNase I may be contaminated. If RNA is degraded without adding DNase I, your RNA sample may be contaminated. A good habit to prevent RNase contamination is to ensure that your gloves or fingers do not touch the inside of the lid and the mouth of the tube when opening and closing the tube containing RNA solution.

5. How should I remove the genomic DNA from my DNA/RNA preparation?

You may add 0.2 units of DNase I to 10-20 ul of DNA/RNA solution in 1x DNase buffer, and incubate at 22-37 °C for 20-30 min. Then run the digested sample in a 0.8% native agarose gel to confirm that the DNA digestion is complete and the RNA bands are discrete. To inactivate the DNase I, use Ambion’s DNase removal reagent or heat-inactivate the DNase I at 65 °C for 15 min.

6. Can I do RT-PCR without removing the contaminating genomic DNA?

DNA removal may be unnecessary if you design and use a 5’ tailed RT primer to make the cDNA and then use a pair of PCR primers, with one of them complementary to the unique tailed region of the RT primer to amplify the cDNA [Hurteau and Spivack. mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts. Anal Biochem. 2002 Aug 15;307(2):304-15; and Chen, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research 2005 33(20):e179].

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 many cells should I seed in each well?

It depends on how fast the cells propagate and how long you need to expose the cells to a test compound. In most cases, it's a good bet to seed 6000-8000 cells/well. After overnight incubation, you will likely have a 20-30% confluent culture to start drug exposure and have ~72 hours of drug exposure period before the no-drug control start to enter apoptosis due to overgrowth. 

3. 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.

4. 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 can use a 570±20nm and 600±20nm filter set.

5. My data does not fit the Four Parameter Model, what can I do?

If you do not include a wide enough range of test concentrations in your experiment or your test compound is too potent or too weak, your experiment may not generate data points around 90-100% or 1-10% viability range. In these cases, the Four Parameter Model, which assumes a sigmoid dose-response curve, may not be able to find the IC50. In such cases, you can either do the experiment again to include enough higher or lower test concentrations or use a theoretical -4 log concentration for 95% viability and/or a +4 log concentration for 5% viability to estimate the IC50 from your existing data points.

1. How should I store the AquaStool kit?

AquaStool may be stored at 4 °C for 12 months. Invert to mix the reagent well before dispensing.

2. Why shouldn’t I use Bleach to disinfect AquaStool preserved fecal specimen?

AquaStool contains guanidine thiocyanate. It may react with Bleach (sodium hypochlorite) and release toxic gases upon mixing if the AquaStool waste volume is sufficiently large. 

3. Can I use AquaStool to extract DNA from other biospecimens?

Yes, for DNA extraction from cultured cells, simply add AquaStool solution to the cell pellet or the culture dish after removing the culture medium, and vortex to lyse the cells. For DNA extraction from animal tissues, such as tail snips, homogenize the tissue sample in AquaStool solution. After lysis and homogenization, follow the fecal DNA extraction protocol to recover the DNA from the cleared lysate.

4. How should I air-dry the fecal samples?

Air-dried fecal samples can be stored long term at room temperature for future genotype verification. To air-dry a mouse fecal pellet, simply incubate the opened microfuge tube containing the fecal pellet on a dry heat bloc at 37 °C for 24 hours.

5. Do I need to ship mouse fecal samples in dry ice?

No, you can ship mouse fecal samples to other laboratories or genotyping facilities at ambient temperature, even in the summer, if they have been air-dried.

6. I had a very weak amplification, any tips?

You may try the following tips to improve fecal DNA amplification and detection. (a) You should re-centrifuge the DNA solution to pellet any insoluble material just before adding it to the PCR reaction, as some insoluble material may develop during storage. (b) You should use 45-65 PCR cycles for the amplification. (c) You may try adding 1 mM DTT to the PCR reaction, which helps re-activate the inactive polymerase. (d) You may try adding 0.1 mg/ml BSA to the PCR reaction, which may sequester residual PCR inhibitors. (e) Finally, a gel imager may be needed to detect the faint amplicon bands.

1. How should I store the AquaPreserve solution?

It may be stored at 4 °C for 12 months. Invert to mix the reagent well before dispensing.

2. When do I need to use ProMelt and ProSink?

ProMelt (Item # 1115) is an ancillary reagent for solubilizing protein pellet precipitated by acetone. It is not needed, if you will not recover the blood proteins. ProSink (Item # 9030) is a protein precipitating solution for DNA/RNA extraction from blood or other nuclease-rich animal tissues. ProSink is optional, if you extract DNA and RNA from bacteria, cultured cells, or plant tissues.

3. How should I thaw 1 ml frozen blood in a 1.5-ml tube?

You could measure 1 ml AquaPreserve to a 15-ml conical tube, then transfer 0.4 ml AquaPreserve to the frozen blood sample, vortex to partially thaw the blood, and quickly transfer the thawed blood back to the conical tube. Repeat until the remaining blood sample is completely thawed.

4. Why didn’t I see the 28S and 18S rRNA bands in the gel?

The 28S and 18S rRNA bands may migrate with the genomic DNA in a native 0.8% agarose gel. However, if you add some salts (e.g., 30 mM NaOAc, pH unadjusted) to the loading dye, you should get a good separation of the 5S, 18S, 28S rRNA, and the genomic DNA bands. Alternatively you can do a DNase I digestion to remove the DNA before running the gel.

5. How should I remove the genomic DNA from the DNA/RNA preparation?

You may add 0.2 units of DNase I to 10-20 ul of DNA/RNA solution in 1x DNase buffer, and incubate at 22-37 °C for 20-30 min. Then run the digested sample in a 0.8% native agarose gel to confirm that the DNA digestion is complete and the RNA bands are discrete. To inactivate the DNase I, use Ambion’s DNase removal reagent or heat-inactivate the DNase I at 65 °C for 15 min.

6. Can I use AquaPreserve to extract total RNA from mouse blood?

7. What’s your suggestion for long-term storage of blood samples?