WET LAB DNA Barcoding: From Samples to Sequences - Northwest ...

WET LAB
CLASS SET
11. Discard the flow-through from your no-cap collection tube and put your spin column back in the no-cap
collection tube.
12. Centrifuge your sample at 10,000 rpm for one minute to remove any residual Wash Buffer from the DNA
binding membrane in the spin column.
[Note: If your centrifuge doesn’t go as fast as 10,000 rpm, centrifuge on maximum speed for 5 minutes.]
13. Label a new 1.7 ml microfuge tube with your name, the date, and your sample name.
14. Place your spin column in the labeled 1.7 ml microfuge tube from Step #13.
15. Add 30 μl of nuclease-free water (labeled “Water”) to the membrane of your spin column, making sure not
to touch the membrane at the bottom of the spin column with your pipette tip.
16. Centrifuge your microfuge tube and spin column at 10,000 rpm for one minute.
[Note: If your centrifuge doesn’t go as fast as 10,000 rpm, centrifuge on maximum speed for 5 minutes.]
17. Discard your spin column.
18. Close the lid of your 1.7 ml microfuge tube with your purified PCR sample and keep the tube on ice, or store it in
the freezer, as instructed by your teacher.
PART II: Preparing Your PCR Sample for DNA Sequencing
The barcoding primers have been specifically designed to work with standard sequencing primers called M13F
(M13 forward) and M13R (M13 reverse), which are provided free of charge by most DNA sequencing facilities.
Many sequencing facilities require specific DNA concentrations, DNA sequencing tubes, and sequencing tube
labeling. Check with your sequencing facility to see what they require.
Determining DNA Concentration:
Using Bioinformatics: Genetic Research
Ideally, DNA concentration is determined using
a spectrophotometer. If a spectrophotometer is
available, ask your teacher for the correct protocol. If a
spectrophotometer is not available, you can estimate your
DNA concentration using your molecular weight standard
from your DNA gel in Lab 3. (See Figure 4.1).
For example, we know from the product information
provided with our molecular weight standard that the
3rd band from the bottom of the gel in Figure 4.1 is 750
base pairs (bp) and contains 25 nanograms (ng) of DNA.
To estimate the amount of DNA present in your PCR
product, you should use the band closest in size to
your PCR product.
For example, if a 700 bp PCR product is approximately
four times as bright as the band at 750 bp in Figure 4.1,
one can estimate that there is about 100 ng of DNA
in the PCR product band (4 x 25 ng = 100 ng).
Figure 4.1: Molecular Weight Standard Used to Calculate
Amount of DNA per Band.
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©Northwest Association for Biomedical Research—Updated October 2012