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DNA extraction
In the interest of preserving vouchers, we extract tissues in two ways.
- For larger specimens we extract a portion of the specimen (e.g. dissected thoracic or leg muscle tissue) and retain the specimen for vouchering. This has the added advantage that additional tissue can be extracted from the same specimen if the original extraction is used up or compromised (e.g contaminated, mislabeled, etc.).
- For smaller specimens, the entire specimen is extracted. The body cavity is opened using dissection or by simply making a slit in an inconspicuous place on the specimen allowing buffer to enter the cavity. Immediately after incubation the portions of the specimen is removed, rinsed and preserved as a voucher. Although not suitable for re-extraction later, one advantage is the complete clearing of the specimen by proteinase-K incubation. Specimens of certain insects (such as Embioptera) are then ideally suited for slide mountiing.
We use the Qiagen DNeasy Blood & Tissue Kit  with individual columns and the protocol for animal tissue. Two major variations are employed:
- We generally incubate overnight.
- We vary the volume of AE buffer used to elute (2 x 200 μl). For smaller specimens, we use a smaller volume (but with two elutions as indicated in the instructions, e.g. 1 x 150 μl and 1 x 100 μl).
Extractions are then checked for success and concentration is determined for optimal dilution by running about 5µl of the raw extraction on a standard 1.5% miniprep agarose gel. A long smear with no obvious high-molecular-weight band probably indicates only short, sheared DNA fragments. A clear high-molecular-weight band indicates good DNA, and its intensity determines what dilution to make for aliquots used for PCR (we often write the desired dilution directly on the vial for future reference). We do not get too specific about dilutions. If high-molecular-weight DNA is abundant in the extraction, we dilute 1:10. If less, then 1:5 or 1:1. Only in rare circumstances do we amplify directly from raw extraction.
We have had some success using Illustra GenomiPhi: Phi29 DNA polymerase-based amplification protocol and then amplifying targeted markers from this product. We have also had limited success sequencing from GenomiPhi product. |
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Kelly B. Miller Lab, Department of Biology, University of New Mexico, 167 Castetter Hall, MSC03 2020, Albuquerque, NM 87131-0001 USA
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