the organism preparation (Table 3) and first two steps of the DNA
isolation (Table 4) can be completed by the instructor before beginning the lab. The methods provided give the students an opportunity to complete the DNA isolation and understand the different
chemicals involved and their roles in the isolation process. Alternatively, a number of DNA isolation kits are available that could be
used to save time, including DNeasy Blood and Tissue kit (Qiagen,
Germantown, Maryland), NucleoSpin Tissue XS Kit (
Macherey-Nagel, Bethlehem, Pennsylvania), and QuickExtract DNA Extraction Solution (Lucigen Corporation, Middleton, Wisconsin).
DNA quantification. The NanoDrop 2000 (Thermo Scientific,
Wilmington, Delaware) spectrophotometer was used for quantification of the DNA. Alternatively, any spectrophotometer with the
ability to measure in the UV light range may be used for quantification. To begin the procedure with the NanoDrop, clean twice with
2 μL of nuclease-free water (NFW) and zeroed with 2 μL NFW.
Measure each sample of DNA twice, using 2 μL of sample. Confirm
that readings are within 10% of each other. The target A260:A280
ratio is 1.7:1.9, but higher ratios may be observed. For the quality
of the DNA, this ratio will reflect the purity of the DNA, since pro-
teins and other contaminants increase the A280 nm and lowering
the ratio of the sample.
Polymerase chain reaction. PCR was run with 1 μL DNA per sample
(assuming between 50 ng/μL and 500 ng/μL concentration). For the
DNA barcoding using the COI locus, primer sequences are provided
in Table 5. A 24 µL of primer and enzyme mix is prepared as shown
in Table 6. The reactions were run on a standard thermocycler using
the conditions shown in Table 7. While the PCR is running, pour an
agarose gel with 1.5 g of agarose powder per 100 mL 1× TAE solution
with 1 μL ethidium bromide added. Alternatively, nontoxic gel stains
such as Sybr-Safe (Thermo Scientific) may be used to reduce waste-handling costs. Allow the gel to set for 15–20 minutes before loading.
Table 3. Instructions for organism preparation, second lab session.
1 Thaw preserved samples.
2 Use disposable razor blade to chop the organism into 6–8 pieces.
3 Place into a 1.5 mL tube labeled with the date of harvest and extraction and organism type.
Table 4. Instructions for DNA extraction and quantification, second lab session.
1 Add 1 μL TNES buffer (50 mM Tris pH 7.5, 0.44 M NaCl, 100 mM EDTA, 0.5% SDS) and 350 μg proteinase K (17.5 μL
of 20 μg/μL ).
2 Incubate at 55°C for 1 hour in a water bath.
3 Add 167 μL saturated 6 M NaCl and shake for 15 seconds.
4 Centrifuge at 13, 200 × g for 30 minutes.
5 Transfer supernatant to clean, labeled micro-centrifuge tube.
6 Add 600 μL of cold 95% ethanol and invert gently to mix.
7 Centrifuge at 13, 200 × g for 30 minutes.
8 Drain supernatant and dispose.
9 Wash with 100 μL of cold 75% ethanol and invert to mix.
10 Centrifuge at 13, 200 × g for 30 minutes.
11 Remove supernatant and allow to air dry.
12 Once dried, add 50 μL of nuclease free water (NFW) and pipette up and down to dissolve the pellet.
Table 5. PCR primer information: macroinvertebrate barcoding primers.a
Primer Name Sequence
LCO1490-Invert COI 5′-TGT AAA ACG ACG GCC AGT CAA CAA ATC ATA AAG ATA TTG G-3′
HC02198-Invert COI 5′-CAG GAA ACA GCT ATG ACT AAA CTT CAG GGT GAC CAA AAA ATC A-3′
a Folmer et al. (1994).