The following are some commonly asked questions
about AquaPlasmidTM,
AquaGenomicTM,
AquaRNATM,
AquaStoolTM,
and AquaBluerTM.
Please read through these questions carefully. The answers
provide additional helpful tips and useful information
for the successful use of AquaPlasmidTM,
AquaGenomicTM,
AquaRNATM,
AquaStoolTM,
and AquaBluerTM.
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) for 12 months. 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) 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 (mtDNA) recovery is desired, you will
need to add Proteinase K to AquaGenomic solution to 100 mg/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. 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. However, if complete RNA removal is
desired, RNase A (200 ug/ml)
can be added to the AquaGenomic solution prior to
extraction.
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
or you are concerned about cross-contamination, you could use the
Proteinase K digestion protocol (see AquaGenomic Tissue Protocol for
details) to extract the DNA.
5. 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.
6. Some of my DNA templates
failed the PCR, what’s wrong?
The
most likely reason is SDS contamination of the DNA prep. If you
rinse the DNA pellet with 70% ethanol from a squirt bottle, be sure
to rinse the entire interior of the tube, including the cap. If the
DNA pellet is large (~10 ul
in size), it may be necessary to re-suspend the pellet in 50 mM
sodium acetate and re-precipitate the DNA in isopropanol to
completely remove any salt and detergent trapped in the
pellet.
7. 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.
8. 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.
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. How should I store the AquaRNA
solution?
It may
be stored at 4-22 °C for 12 months. Vortex to mix the solution well
before dispensing.
2. Do I need to add RNase
Inhibitor to the final RNA solution?
No. AquaRNA is very
effective at inactivating and removing RNases. You could even
leave your RNA samples at room temperatures for days without
noticing any RNA degradation.
3. How should I remove DNA from
the RNA preparation?
If DNA removal is
desired, you may use 0.2 units of RNase-free DNase I to
treat 10-20 ul of DNA/RNA preparation at 22-37 °C for 30
min. The DNase I can then be inactivated by heat-inactivation at 65
°C for 15 min or be removed with Ambion’s DNase Removal
Reagent. However, DNA removal may be 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)].
4. 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 inactivate RNases to improve the
RNA yield.
5. I did not see the 28S and 18S
rRNA bands in the gel, why?
The
28S and 18S rRNA bands may migrate with genomic DNA in a
non-denaturing gel. To see the RNA bands, you may digest the DNA/RNA
prep with DNase I, and then run it alongside an undigested control
in a non-denaturing 0.6-0.7% agarose gel.
6. The A260/A230 ratio of my
DNA/RNA prep is very low, what’s the problem?
This
may be caused by guanidine thiocyanate contamination. You may
re-precipitate the DNA/RNA in 50 mM sodium acetate with 1 vol of
isopropanol to clean up the contaminated DNA/RNA. You could prevent
guanidine thiocyanate contamination, if you do not use more than 1.5
vol of isopropanol to precipitate the DNA/RNA from 1 vol of cell
lysate, do not incubate the isopropanol/lysate mixture on ice before
pelleting the DNA/RNA, and rinse the DNA/RNA pellet and its tube
thoroughly, including the cap as it may catch the reagent as you
decant to discard the isopropanol supernatant.
7. My RNA was degraded, where was
the RNase coming from?
To
troubleshooting RNase contamination, you may set up a DNase I
digestion in 1x DNase I buffer. Before adding DNase I, divide the
sample into two and add DNase I to only one of the two. If RNA loss
is seen only in the DNase I treated sample, it indicates that
the DNase I is contaminated. If RNA is lost in both samples,
you have RNase contamination of your RNA
prep.
8. 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.
AquaStool
FAQ
1. How should I store the AquaStool
kit?
It may be
stored at 4-22 °C for 12 months. Vortex to mix the reagent well
before dispensing.
2. Can I scale down the volume of
AquaStool for extraction?
Of course, AquaStool
extraction protocol is scalable. If your biomarker is abundant, such
as bacterial DNA and RNA, you may use 0.25 ml of AquaStool to
extract 25 mg of feces.
3. Why shouldn't I use Bleach to
disinfect AquaStool preserved fecal specimen?
AquaStool contains
guanidine thiocyanate (GITC). It may react with Bleach, i.e., sodium
hypochlorite (NaClO), and release toxic gases, upon mixing.
4. Can I use AquaStool to extract DNA and
RNA from other biospecimens?
Yes, for extraction
from cultured cells, you simply add AquaStool solution to the cell
pellet or the culture dish after removing the culture medium and
vortex the resulting cell lysate to extract DNA/RNA. For animal or
plant tissues, you could homogenize the tissue in AquaStool solution
to extract DNA/RNA. Not sonication is needed.
5. Do I need to prepare 70% ethanol with
commercial nuclease-free water?
No, deionized water
from laboratory water purification systems is nuclease-free. Most
RNA sample degradation is due to endogenous RNase contamination and
AquaStool is very effective at inactivating and removing endogenous
RNases.
6. Can I use AquaStool to extract fecal
DNA for transgenic mouse genotyping?
Yes, unlike tail
clipping, fecal DNA genotyping is noninvasive and repeated sampling
is allowed. Furthermore, you could use RNAtyping to screen
for animals that not only carry the desired transgene but
also express the transgene product. This is a unique and useful
feature of concurrent fecal or cell or tissue DNA/RNA extraction
with AquaStool.
7. How should I store the mouse
fecal samples?
It’s a good
idea to collect 2-4 fecal pellets from each animal, extract DNA/RNA
from only one pellet, and store the remaining pellets for possible
future extraction. The fecal pellets may be stored at 4-22 °C for
months until DNA extraction, however, for fecal RNA extraction, the
fecal pellets should be stored at –80
°C.
8. 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 0.8% native agarose gel by electrophoresis).
AquaPreserve FAQ
1. How should I store the AquaPreserve
kit?
It may be stored at
4-22 °C for 12 months. Vortex 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 the protein pellet
precipitated by acetone. You do not need to buy ProMelt if you do
not need to recover proteins. ProSink (Item # 9015) is a protein
precipitating solution; it is used to precipitate hemoglobin and
other proteins when extracting DNA/RNA from whole blood. You do not
need to buy ProSink if you only use AquaPreserve to extract DNA and
RNA from cultured cells or solid tissue
specimens.
3. Can I extract RNA from ACD blood with
AquaPreserve?
Yes, but the RNA
yield is only about 1/5th of that extracted from EDTA
blood.
4. I did not see the 28S and 18S rRNA
bands in the gel, why?
The 28S and 18S rRNA
bands may migrate with genomic DNA in non-denaturing agarose gel. To
see the RNA bands, you may digest the DNA/RNA prep with DNase I and
then run the digested sample alongside an undigested control in a
non-denaturing 0.7% agarose gel.
5. How should I remove the genomic DNA
from the preparation?
If DNA removal is
desired, you may use 0.2 units of DNase I to digest 10-20 ul of
DNA/RNA prep at 22 °C for 30 min. Following the incubation, you can
run a non-denaturing 0.7% agarose gel to examine the completion of
the digestion. The DNase I can be heat-inactivated at 65 °C for 15
min.
6. The A260/A230 ratio of my DNA/RNA prep
was very low, what could be the problem?
This may be caused
by guanidine thiocyanate contamination. Make sure you do not use
more than 1.5 vol of isopropanol to precipitate the DNA/RNA from 1
vol of lysate, do not incubate the isopropanol/lysate mixture on ice
before pelleting the DNA/RNA, and rinse the DNA/RNA pellet and its
tube thoroughly. You may re-precipitate the DNA/RNA in 50 mM sodium
acetate with 1 vol of isopropanol to clean up the contaminated
DNA/RNA.
7. Can I use this AquaPreserve Blood
Protocol to extract total RNA from mouse blood?
Yes, mouse blood
contains abundant of RNA. For human blood, the RNA yield is only
~3-5 ug/ml of blood, but for mouse blood, the RNA yield could be
~50-80 ug/ml of blood. Therefore, you may scale down to use 50-100
ul of mouse whole blood for RNA extraction.
8. Why shouldn't I use Bleach to
disinfect AquaPreserve preserved specimens?
AquaPreserve
contains guanidine thiocyanate (GITC). It may react with Bleach,
i.e., sodium hypochlorite (NaClO), and release toxic gases, upon
mixing.
