DNAPurificationfromBloodorBodyFluids

實驗概要

試劑盒，操作簡單，稍有點貴，還可以接受。

實驗材料

blood

實驗步驟

Protocol: DNA Purification from Blood or Body Fluids
(Spin Protocol)
This protocol is for purification of total (genomic, mitochondrial, and viral) DNA from
whole blood, plasma, serum, buffy coat, lymphocytes, and body fluids using a
microcentrifuge. For total DNA purification using a vacuum manifold, see “Protocol:
DNA Purification from Blood or Body Fluids (Vacuum Protocol)” on page 30.
Important points before starting
■ All centrifugation steps are carried out at room temperature (15–25°C).
■ Use carrier DNA if the sample contains <10,000 genome equivalents (see
page 18).
■ 200 μl of whole blood yields 3–12 μg of DNA. Preparation of buffy coat (see
page 19) is recommended if a higher yield is required.
Things to do before starting
■ Equilibrate samples to room temperature.
■ Heat a water bath or heating block to 56°C for use in step 4.
■ Equilibrate Buffer AE or distilled water to room temperature for elution in step 11.
■ Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared
according to the instructions on page 17.
■ If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.
Procedure
1.  Pipet 20 μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml
microcentrifuge tube.
2.  Add 200 μl sample to the microcentrifuge tube. Use up to 200 μl whole blood,
plasma, serum, buffy coat, or body fluids, or up to 5 x 10⁶ lymphocytes in 200 μl PBS.
If the sample volume is less than 200 μl, add the appropriate volume of PBS.
QIAamp Mini spin columns copurify RNA and DNA when both are present in
the sample. RNA may inhibit some downstream enzymatic reactions, but not PCR.
If RNA-free genomic DNA is required, 4 μl of an RNase A stock solution
(100 mg/ml) should be added to the sample before addition of Buffer AL.
Note: It is possible to add QIAGEN Protease (or proteinase K) to samples that have
already been dispensed into microcentrifuge tubes. In this case, it is important to
ensure proper mixing after adding the enzyme.
3.  Add 200 μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.
In order to ensure efficient lysis, it is essential that the sample and Buffer AL are
mixed thoroughly to yield a homogeneous solution.
If the sample volume is larger than 200 μl, increase the amount of QIAGEN
Protease (or proteinase K) and Buffer AL proportionally; for example, a 400 μl
sample will require 40 μl QIAGEN Protease (or proteinase K) and 400 μl Buffer
AL. If sample volumes larger than 400 μl are required, use of QIAamp DNA Blood
Midi or Maxi Kits is recommended; these can process up to 2 ml or up to 10 ml
of sample, respectively.
Note: Do not add QIAGEN Protease or proteinase K directly to Buffer AL.
4.  Incubate at 56°C for 10 min.
DNA yield reaches a maximum after lysis for 10 min at 56°C. Longer incubation
times have no effect on yield or quality of the purified DNA.
5.  Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside
of the lid.
6.  Add 200 μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing
for 15 s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove
drops from the inside of the lid.
If the sample volume is greater than 200 μl, increase the amount of ethanol
proportionally; for example, a 400 μl sample will require 400 μl of ethanol.
7.  Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2 ml
collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g
(8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection
tube (provided), and discard the tube containing the filtrate.*
Close each spin column in order to avoid aerosol formation during centrifugation.
Centrifugation is performed at 6000 x g (8000 rpm) in order to reduce noise.
Centrifugation at full speed will not affect the yield or purity of the DNA. If the
lysate has not completely passed through the column after centrifugation,
centrifuge again at higher speed until the QIAamp Mini spin column is empty.
Note: When preparing DNA from buffy coat or lymphocytes, centrifugation at full
speed is recommended to avoid clogging.
8.  Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1 without
wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min.
Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and
discard the collection tube containing the filtrate.*
It is not necessary to increase the volume of Buffer AW1 if the original sample
volume is larger than 200 μl.
* Flow-through contains Buffer AL or Buffer AW1 and is therefore not compatible with bleach. See page 8 for
safety information.
9.  Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2
without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g;
14,000 rpm) for 3 min.
10. Recommended: Place the QIAamp Mini spin column in a new 2 ml collection tube
(not provided) and discard the old collection tube with the filtrate. Centrifuge at full
speed for 1 min.
This step helps to eliminate the chance of possible Buffer AW2 carryover.
11.  Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube (not
provided), and discard the collection tube containing the filtrate. Carefully open
the QIAamp Mini spin column and add 200 μl Buffer AE or distilled water.
Incubate at room temperature (15–25°C) for 1 min, and then centrifuge at
6000 x g (8000 rpm) for 1 min.
Incubating the QIAamp Mini spin column loaded with Buffer AE or water for 5 min
at room temperature before centrifugation generally increases DNA yield.
A second elution step with a further 200 μl Buffer AE will increase yields by up
to 15%.
Volumes of more than 200 μl should not be eluted into a 1.5 ml microcentrifuge
tube because the spin column will come into contact with the eluate, leading to
possible aerosol formation during centrifugation.
Elution with volumes of less than 200 μl increases the final DNA concentration in
the eluate significantly, but slightly reduces the overall DNA yield (see Table 5,
page 26). For samples containing less than 1 μg of DNA, elution in 50 μl Buffer
AE or water is recommended. Eluting with 2 x 100 μl instead of 1 x 200 μl does
not increase elution efficiency.
For long-term storage of DNA, eluting in Buffer AE and storing at –20°C is
recommended, since DNA stored in water is subject to acid hydrolysis.
A 200 μl sample of whole human blood (approximately 5 x 10⁶ leukocytes/ml)
typically yields 6 μg of DNA in 200 μl water (30 ng/μl) with an  A260/A280 ratio of
1.7–1.9.
For more information about elution and how to determine DNA yield, purity, and
length, refer to pages 25–26 and Appendix A, page 51

附    件   (共1個附件,占1.07M)

Isolation_of_g

1.07M

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