FAQ - www.aquaplasmid.com

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.

AquaPlasmid FAQ

AquaGenomic FAQ

AquaRNA 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, neither AquaLysis nor AquaPlasmid solution contains RNase A. However, for complete RNA removal, you may use RNase A containing buffer (such as Qiagen's P1 buffer) in stead of water to suspend the cell pellet.

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 especially for miniprep. 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 once. For maxiprep, you may use centrifugation to re-pellet the DNA with 70% ethanol for a better rinse.

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.

AquaGenomic FAQ

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, for DNA extraction from dry blood spot, saliva, and some fibrous tissues, or if mitochondrial DNA recovery is desired, you may add Proteinase K to AquaGenomic solution to 50 ug/ml (e.g., add 1 ul of 5 mg/ml Proteinase K to 100 ul of AquaGenomic) and incubate the samples at 60 °C for 60 min and then at 95 for 10 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.

AquaRNA FAQ

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 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].

AquaStool FAQ

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, 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.

AquaPreserve FAQ

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 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.

	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. AquaPlasmid FAQ AquaGenomic FAQ AquaRNA 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, neither AquaLysis nor AquaPlasmid solution contains RNase A. However, for complete RNA removal, you may use RNase A containing buffer (such as Qiagen's P1 buffer) in stead of water to suspend the cell pellet. 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 especially for miniprep. 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 once. For maxiprep, you may use centrifugation to re-pellet the DNA with 70% ethanol for a better rinse. 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.
	AquaGenomic FAQ 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, for DNA extraction from dry blood spot, saliva, and some fibrous tissues, or if mitochondrial DNA recovery is desired, you may add Proteinase K to AquaGenomic solution to 50 ug/ml (e.g., add 1 ul of 5 mg/ml Proteinase K to 100 ul of AquaGenomic) and incubate the samples at 60 °C for 60 min and then at 95 for 10 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.
	AquaRNA FAQ 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 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].
	AquaStool FAQ 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, 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.
	AquaPreserve FAQ 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 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.
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