No, AquaGenomic Solution is stable at
room temperature (~22° C). If precipitates appear due to cold
weather, incubating at 37° C for 5-10 minutes will clear the
solution.
2. Should I keep the solutions
and samples on ice while carrying out the isolation?
No. Most of the isolation steps should be
performed at room temperature (~22° C). For DNA isolation from
animal cells, cell lysis at 60° C may increase the yield by ~25%. For
DNA isolation from cells with a cell wall, such as bacterial and
plant cells, the cell lysis should be carried out at
60-90° C for 15-30 minutes.
3. Does
AquaGenomic Solution contain Proteinase K?
No. AquaGenomic Solutions can be used to
extract DNA from cells and tissues without needing protease
digestion. This reduces the time required for genomic DNA isolation
from solid tissues from a full day to only ~15 minutes.
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, you can smash the tissue with a pair of tweezers. Another
option is to use the 60° C extraction method, which does not
require a homogenizer but a few hours of 60° C incubation. At the end of incubation,
you may add some glass or ceramic beads to the sample and vortex
vigorously to disrupt the tissue.
5. 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.
6. 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.
7. 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.
8. 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.
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