FREQUENTLY ASKED QUESTIONS
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.
1. Do I need to store the AquaPlasmid kit at 4 or –20 °C?
No, AquaPlasmid solution should be stored at room temperature (~22 °C). If the temperature is below 18 °C, such as during transit or storage in the winter, precipitation may develop. If so, incubate the solution at 37-55 °C for a few minutes to re-solubilize it before use.
2. How does AquaPlasmid work?
AquaPlasmid combines the functions of traditional P1 buffer (cell suspension), P2 buffer (cell lysis), N3 buffer (debris removal), and silica column (plasmid DNA purification) in a single solution. It lyses the cells without denaturing the DNA, extracts the plasmid DNA while keeps other cellular contaminants in the cell debris. The plasmid DNA is subsequently precipitated from the clear lysate with isopropanol.
3. Does AquaPlasmid contain guanidine salts?
No, AquaPlasmid does not contain guanidine salts. They are required for column-based purification and not biodegradable. When they are mixed with Bleach on the floors, in the sinks or sewage, toxic and mutagenic fumes may be released into the environment, which could be harmful to the workers and aquatic lives.
4. How should I scale up and down the reagents for other culture volumes?
We recommend 200 ul of AquaPlasmid for each 1.5 ml of overnight culture. However, you may use 150 ul of AquaPlasmid for each 1.5 ml of overnight LB culture without noticeable difference in plasmid DNA yield. On the other hand, if the cell density is too high, such as in overnight TB culture, it may result in incomplete cell lysis and debris removal, and you will need to use 300 ul of AquaPlasmid for each 1.5 ml culture.
5. Any specific recommendation on the use of the AquaPlasmid method?
We recommend that you use the AquaPlasmid method only on E. coli strains that carry the endA1 mutation, such as TOP10, DH5a, XL-1 Blue, JM109, and SURE, etc. If the genotype is unknown, you should incubate an aliquot of the purified DNA in a restriction enzyme buffer at 37 °C for 12 hours to confirm there is no DNA degradation by gel electrophoresis. If the DNA is degraded, you may try incubating the purified DNA at 75-85 °C for 20-30 min to inactivate the contaminating nucleases.
1. Do I need to keep AquaGenomic in the freezer?
No, AquaGenomic Solution is stable at room temperature (~22 °C) for >1 year.
2. Does AquaGenomic Solution contain Proteinase K?
No. AquaGenomic can be used to extract DNA from most cells and tissues without needing protease digestion. However, adding Proteinase K (50 ug/ml) to AquaGenomic solution can increase DNA yield and is required for mitochondrial DNA extraction. You may homogenize the sample in Proteinase K containing AquaGenomic, incubate it at 56-60 °C for 1-2 hrs and then at 95 °C for 10-15 min to inactivate the Proteinase K.
3. AquaGenomic sounds like a “green” product, any particular cautions?
AquaGenomic is nontoxic and non-corrosive. It contains no phenol, chloroform, guanidine HCl, and other harmful chemicals commonly used for DNA extraction. There is no particular precaution while using AquaGenomic; you just need to follow standard good laboratory practice in handling laboratory chemicals.
4. I am worried about cross-contamination using homogenizers, any tips?
Between uses, you may 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 DNA cross-contamination. If you still feel uneasy, you may use Proteinase K digestion to disrupt the tissues without using a homogenizer, or use a multichannel bead beater for homogenization in screw-capped tubes.
5. Do I have to use the lysate immediately for PCR?
No, you may store the lysate at 4 °C until analysis. If the lysate has been incubated at 85 °C for 20 min, it may even be left at room temperature until analysis.
6. I got a weak PCR amplification using the lysate, how can I improve it?
You may try a few things to optimize the amplification: a) try use different amount of lysate for the PCR, form 0.25 ul undiluted lysate to 20x diluted lysate, b) add 0.1 mg/ml BSA to the PCR reaction, c) add 1 mM DTT to the PCR reaction, and d) increase the PCR cycle number to 45 cycles.
1. How should I store the AquaRNA solution?
AquaRNA may be stored at 22 °C for 12 months. If AquaRNA becomes precipitated when exposed to low temperatures, you may incubate it at 37 °C for 15-20 min to resolubilize.
2. Do I need to use ProMelt and ProSink?
ProMelt (Item # 1115) is not needed, if you don't recover proteins. ProSink (Item # 9030) is not required for DNA/RNA extraction from bacteria, cultured cells, and most plant and animal 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 uM 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 use DNase I 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. How should I store the AquaStool kit?
AquaStool may be stored at 22 °C for 12 months. If AquaStool becomes precipitated when exposed to low temperatures, you may incubate it at 37 °C for 15-20 min to resolubilize.
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, how can I improve it?
You may try the following tips to improve fecal DNA amplification and detection. (a) Avoid taking up fecal debris when transferring the clear lysate. (b) Re-centrifuge the DNA solution to pellet any insoluble material just before adding it to the PCR reaction. (b) Use 45-65 PCR cycles for the amplification. (c) Add 1 mM DTT to the PCR reaction, which helps re-activate the inactive polymerase. (d) Add 0.1 mg/ml BSA to the PCR reaction, which may sequester residual PCR inhibitors. (e) Use a gel imager to visualize faint amplicon bands. (h) Re-do the extraction and double the amount of AquaStool used in the extraction.
1. How should I store the AquaPreserve solution?
AquaPreserve may be stored at 22 °C for 12 months. If AquaPreserve becomes precipitated when exposed to low temperatures, you may incubate it at 37 °C for 15-20 min to resolubilize.
2. When do I need to use ProMelt and ProSink?
ProMelt (Item # 1115) is not needed, if you will not recover the proteins. ProSink (Item # 9030) is a protein-precipitating reagent and it is required for blood DNA/RNA extraction.
3. How should I thaw 1 ml frozen blood in a 1.5-ml tube?
Ideally, the fresh blood sample is aliquoted in tubes 3x of the sample volume, or pre-mixed with AquaPreserve and ProSink prior to freezing. However, to process existing 1-ml frozen blood sample in a 1.5-ml tube, you may either cut open the tube to retrieve the frozen blood pellet or use 0.4 ml of AquaPreserve to partially thaw the frozen blood repeatedly and transfer it to a large tube.
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. If you add some salts (e.g., 30 uM NaOAc, pH unadjusted) to the loading dye, you may get a better separation of the DNA and RNA bands. However, it would be better to 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 U 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. To inactivate the DNase I, you may use Ambion’s DNase removal reagent or inactivate the DNase I at 65 °C for 15 min.
6. Can I do RT-PCR without removing the contaminating genomic DNA?
Complete DNA removal may be difficult to achieve and unnecessary if you use intron-spanning primers for the PCR amplification. You may also 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], especially when intron-spanning is unavailable. In any case, you should always include a no-RT control in your amplification to confirm that your primers do not amplify the contaminating genomic DNA.
7. Can I use AquaPreserve to extract total RNA from mouse blood?
Yes, mouse blood contains an abundance of RNA. RNA yield is only 1-2 ug/ml of human blood, but RNA yield can be 50-80 ug/ml of mouse blood.
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 repeat the experiment 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.
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