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?
It may be stored at 22 °C for 12 months.
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.
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. And finally (e) use a gel imager to visualize faint amplicon bands.
1. How should I store the AquaPreserve solution?
It may be stored at 22 °C for 12 months.
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. How should I store the AquaMutant kit?
AquaMutant, its Start and Stop solutions may be stored at room temperature for 12 months.
2. How does AquaMutant work?
AquaMutant is a proprietary formulation of mutagenic chemicals, which may be activated upon mixing with the Start solution. The activated AquaMutant can modify the bases of DNA to cause base substitutions during DNA synthesis in vitro and in vivo.
3. What types of mutation does AquaMutant introduce?
AquaMutant primarily modifies A, C, and G, causing base transitions (50-60%) and transversions (30-40%). The mutation rate may be controlled by varying the concentration of activated AquaMutant and its exposure time. Direct sequencing of plasmid DNA treated with 100% activated AquaMutant at 37 °C for 24 hours shows substitutions at nearly every nucleotide positions.
4. What precautions should I take using AquaMutant?
WARNING: ACTIVATED AQUAMUTANT IS A STRONG MUTAGEN. You must wear protective lab coat, gloves, masks and goggles to prevent skin contact or ingestion of the reagent! Activated AquaMutant can be readily destroyed by autoclave. It can also be quenched and inactivated by mixing with bacterial culture media, such as LB medium. Unactivated AquaMutant solution should be disposed as hazardous waste in accordance with local regulations.
5. What type of study may be classified as DURC research?
While selections or evolutions of dangerous mutants or pathogens, such as making them drug-resistant, vaccine-resistant, more virulent, transmissible, or having a broader host range, etc., are necessary for understanding their mechanisms and developing countermeasures, they could be misused, therefore, are dual-use research of concern (DURC). You must seek pertinent permits and carry out DURC studies in appropriate biosafety facilities. You should handle microorganism mutant libraries as biohazards and not release them into the environment.
6. My random mutagenesis didn’t work, what may be the causes?
If your random mutagenesis failed, it is likely that the host (e.g., TOP10 E. coli strain) may be able to repair the AquaMutant modified nucleobases or the mismatches. You may either first amplify the mutated plasmid by PCR or transform it into a MutS E. coli strain, such as BMH 71-18 to amplify the library before its selection in the host strains of interest.
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