Freshwater Zooplankton barcode with special reference to Copepoda and cyclopoida

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Permalink Reply by Manuel Elias-Gutierrez on November 29, 2010 at 10:23am

Hi Pranay

Is not easy to work on freshwater zooplankton. We extract with a Hotshot method (see Montero-Pau, J., A. Gomez & J. Munoz, 2008. Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs, Limnology and Oceanography-Methods 6: 218-222) with a final centrfugation at 10k RPM for 5 min (with more than 90% success to extract DNA).

Then amplification is not easy, I get 30-40% success (see Elías-Gutiérrez, M., F. Martínez-Jerónimo, N. V. Ivanova & M. Valdez-Moreno, 2008. DNA barcodes for Cladocera and Copepoda from Mexico and Guatemala, highlights and new discoveries, Zootaxa 1849: 1-42). We are working with primers right now.
Protocols we use are in this paper.

Currently, we are getting more success with rotifers than microcrustaceans.

Regards
Manuel

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Permalink Reply by Pranay Sharma on December 6, 2010 at 1:07am

Good day Manuel,

Thank you for your advice, I have developed certain primers ( some of them are redundant/degenerate primers and LCO series 1 to 4) these primers have successfully amplified the COI region for most Daphnia species(image attached) using 2 stage amplification Thermocycle profile,but same can't be said for copepods especially for family cyclopida ( any advice will be helpful) .

The protocols for DNA extraction mentioned earlier ion paper was found to be less successful for my study and hence have to modify the Qiagen extraction protocol; which is costly but has Success rate of 95% for even 10 year old ethanol preserved sample.

It would be pleasure if we share the methodology and if worthwhile can jointly publish the work.

Regards
Pranay Sharma

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