practical part of
Molecular
Diagnostics
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Lab.1
DNA extraction from blood
Objectives:
1- DNA extraction from blood according to the promega kit.
2- DNA Analysis by Agarose Gel Electrophoresis.
3- Determination of DNA concentration by Spectrophotometer.
Description:
The Wizard Genomic DNA Purification Kit (Promega company Cat. No.
A1120) is designed for isolation of DNA from white blood cells. The Wizard
Genomic DNA Purification Kit is based on a four-step process.
The first step in the purification procedure lyses the cells and the nuclei. For
isolation of DNA from white blood cells, this step involves lysis of the red
blood cells in the Cell Lysis Solution, followed by lysis of the white blood cells
and their nuclei in the Nuclei Lysis Solution. An RNase digestion step may be
included at this time; it is optional for some applications. The cellular proteins
are then removed by a salt precipitation step, which precipitates the proteins
but leaves the high molecular weight genomic DNA in solution. Finally, the
genomic DNA is concentrated and desalted by isopropanol precipitation.
DNA purified with this system is suitable for a variety of applications, including
amplification, digestion with restriction endonucleases and membrane
hybridizations.
Isolating Genomic DNA from Whole Blood (300μl):
1- Add 900μl of Cell Lysis Solution to a sterile 1.5ml microcentrifuge tube.
2- Gently rock the tube of blood until thoroughly mixed; then transfer 300 μl
blood to the tube containing the Cell Lysis Solution. Invert the tube 5–6 times
to mix.
3- Incubate the mixture for 10 minutes at room temperature (invert 2–3 times
once during the incubation) to lyse the red blood cells. Centrifuge at 13,000–
16,000 × g for 20 seconds at room temperature.
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4- Remove and discard as much supernatant as possible without disturbing
the visible white pellet. Approximately 10–20μl of residual liquid will remain in
the 1.5ml tube.
5. Vortex the tube vigorously until the white blood cells are resuspended (10–
15 seconds).
6. Add 300μl Nuclei Lysis Solution to the tube containing the resuspended
cells. Pipet the solution 5–6 times to lyse the white blood cells. The solution
should become very viscous.
7. Optional: Add 1.5μl RNase Solution to the nuclear lysate, and mix the
sample by inverting the tube 2–5 times. Incubate the mixture at 37°C for 15
minutes, and then cool to room temperature.
8- Add 100μl Protein Precipitation Solution to the nuclear lysate, and vortex
vigorously for 10–20 seconds. Small protein clumps may be visible after
vortexing.
9- Centrifuge at 13,000–16,000 × g for 3 minutes at room temperature. A dark
brown protein pellet should be visible.
10- Transfer the supernatant to a clean 1.5ml microcentrifuge tube containing
300μl of room-temperature isopropanol.
11- Gently mix the solution by inversion until the white thread-like strands of
DNA form a visible mass.
12- Centrifuge at 13,000–16,000 × g for 1 minute at room temperature. The
DNA will be visible as a small white pellet.
13- Decant the supernatant, and add one sample volume of room temperature
70% ethanol to the DNA. Gently invert the tube several times to wash the DNA
pellet and the sides of the microcentrifuge tube. Centrifuge as in Step 12.
14. Carefully aspirate the ethanol using either a drawn Pasteur pipette or a
sequencing pipette tip. The DNA pellet is very loose at this point and care
must be used to avoid aspirating the pellet into the pipette. Invert the tube on
clean absorbent paper and air-dry the pellet for 10–15 minutes.
15. Add 100μl DNA Rehydration Solution to the tube and rehydrate the DNA
by incubating at 65°C for 1 hour. Periodically mix the solution by gently
tapping the tube.
Alternatively, rehydrate the DNA by incubating the solution overnight at room
temperature or at 4°C.
16. Store the DNA at 2–8°C.
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DNA Quality and quantity check using agarose gel:
To test for the quality and amount of isolated DNA, a sample of isolated DNA
(5 µL) is run on the 1% agarose gel in TBE buffer or TAE buffer to determine
if the DNA is degraded. Look for a tight band of high molecular weight;
smearing would indicate degraded DNA.
Place 5 µL of your DNA in a microfuge tube, add 5 µL of water and 2 µL of
loading dye and place all the solution into one of the wells in the gel. In one
lane place a DNA concentration standard (usually a known amount of lambda
DNA). A range of lambda DNA amounts (50ng, 100ng, 250ng and 500ng) can
be used to get a more accurate estimation of the DNA concentration. We will
use these 4 concentration standards
Run the gel at 100V for 90 minutes
Determine the DNA quality on a UV-transilluminator, photograph if possible
and determine the DNA concentration of samples visually using the λ DNA
amounts for comparison.
Dilute an aliquot of your DNAs to a concentration of 10ng/µL for the PCR
reactions.
Determination of DNA concentration by Spectrophotometric Estimation:
When working with DNA, it is often important to know its concentration (recall
that concentration will be expressed in units of mass per volume). The easiest
means of determining DNA concentration is through spectrophotometric
analysis.
Rule: The concentration of pure double-stranded DNA with an A260 of 1.0 is
50 g/ml. Thus, one can use the following formula to determine the DNA
concentration of a solution.
Unknown (g/ml)/ Measured A260 = 50 (g/ml)/ 1.0 A260
Since there is a linear relationship between absorbance and DNA
concentration, we can use some simple algebra and reformulate as follows:
Unknown g/ml = 50 g/ml x Measured A260 x dilution
factor (see below)
1. Obtain two falcon tubes and label one 'B' (for blank) and the other 'D' (for
DNA).
2. Add 2 ml of TE buffer to the B' tube and 1.99 ml of TE buffer to the 'D' tube.
3. Add 10 l of your DNA solution to the 'D' tube. Mix by gentle pipeting.
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4. Transfer the entire contents of the B tube to a glass cuvette in the proper
slot of the spectrophotometer (which is set at A260). Since there is no DNA in
this tube, the reading should be close to 0. If not, set the spectrophotometer
so it reads 0. You have now calibrated the machine.
5. Using a plastic pipet, remove the water from the B tube and immediately
replace it with the contents of 'D' tube. Wait several seconds and allow the
A260 reading to stop drifting. When the reading becomes stable, write down
the A260. This is the value you will use in the above formula.
The dilution factor
We took 10 l of the DNA solution and diluted it to a final volume of 2 ml
(2000 l). Thus, we diluted it by a factor of 0.005. When you measured the
DNA concentration, you obtained an absorbance value of the diluted solution,
but you want to know the concentration of your original solution (the one you
would work from when doing experiments). To do this, you must multiply your
determined concentration by the reciprocal of this dilution factor (1/0.005). Or,
to put it simply, just use the following formula:
Unknown g/ml = A260 x 50 g/ml x 200
In contrast to nucleic acids, proteins have a UV absorption maximum of 280
nm. The absorbance of a DNA sample at 280 nm gives an estimate of the
protein contamination of the sample. The ratio of the absorbance at 260 nm /
absorbance at 280 nm is a measure of the purity of a DNA sample; it should
be between 1.7 and 2.
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