DMSO/EDTA/NaCl protocol

References

Dawson, Mike N., Raskoff, Kevin A., and Jacobs, David K. (1998) Field preservation of marine invertebrate tissue for DNA analyses. Molecular Marine Biology and Biotechnology 7(2): 145-152.

Dillon, N., Austin, A.D., and Bartowsky, E. (1996) Comparison of preservation techniques for DNA extraction from hymenopterus insects. Insect Molecular Biology 5(1): 21-24.

Kilpatrick, C. William. (2002) Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods. Biochemical Genetics 40(1/2): 53-62.

Seutin, G., White, B.N., Boag, P.T. (1991) Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology-Revue Canadienne de Zoologie 69 (1): 82-90.

Recipe

0.25M EDTA pH 7.5

20% DMSO

NaCl saturated

1. Measure out 23.265g of EDTA disodium salt with FW 372.24 for a 250ml solution. (This may be different depending on the FW of your EDTA salt). Add 50ml of deionized water to the EDTA salt and stir.

**Make sure to sure EDTA disodium salt otherwise more NaOH is needed to pH the EDTA.

2. Make 1M NaOH to pH the EDTA. The EDTA should be around a pH of 3 or 4 to begin with. It will take roughly 50ml of the 1M NaOH to pH the EDTA to 7.5. The EDTA will then begin to dissolve slowly. Be patient. Heating to 30°C helps.

3. Once all the EDTA salt is dissolved bring the volume up to 200ml with deionized water. Then add the 20% DMSO, which is 50ml for a 250ml solution. Return to a beaker and stir for a few minutes.

4. Add NaCl until it no longer dissolves. Pour the solution into a bottle leaving most of the salt crystals in the beaker.

** Catalog numbers of materials if ordering from Fisher:

DMSO 500ml D128-500 $20.51( UCR discount)

EDTA, disodium salt 500g S312-500 $61.88 (UCR discount)

Sodium Chloride granular 500g S640-500 $12.29 (UCR discount)

Preserving samples

1. Extract nematodes and concentrate them into water (or sea water) suspension using your method of choice. Check under a dissecting microscope to make sure that the extraction worked and provided you with plenty of live nematodes. The nematodes are now ready for preservation.

2. Take your sample, pour it through a #500 mesh sieve (25um pores) and concentrate the nematodes at the bottom of the sieve. Try to drain as much of the water (or sea water) in the sieve as possible without allowing the worms to desiccate.

3. Immediately add DMSO solution to the side of the sieve where the nematodes are concentrated. Tilt the sieve with DMSO over a vial and rinse the mesh with DMSO until all the soil, nematodes etc are washed into the vial. Label vial and store at room temperature or in a refrigerator.

**Examination of nematodes after being placed in the DMSO solution is that they shrivel slightly and return to their original form after being in the solution about 15 minutes.

Extracting samples from DMSO

1. Pick the nematodes you want from the DMSO solution and place in a dish with water (sea water) for a few minutes to remove any salt or DMSO that might be attached.

PCR Results

PCR was performed on the D2D3 region for each nematode

**Smearing is due to using too much Taq in the PCR reaction.

Morphological Results

5M7G5 from Kern Co. sample that was placed in DMSO 5/13/05 and capture 7/7/05.

3M7G5 is from Saharna, Moldova was placed in DMSO 6/28/05 and was captured 7/7/05.

4W5G5 is from the Black Sea was placed in DMSO 6/25/05 and was captured 7/5/05.

8M2F5 is from the UCR Botanic Gardens was placed in DMSO 5/13/05 and was captured 6/2/05.

Making Permanent Mounts

1. Wash the sample in the DMSO solution on a #500 mesh sieve with DI water until all the NaCl, DMSO, and EDTA is removed.

2. Place the sample in a shallow cavity block or embryo dish. Place the cavity block in an airtight container with half an inch of 96% ethanol surrounding the cavity block. Place the airtight container in an oven set to 40C overnight for about 12 hours.

3. Remove the cavity block the next morning and cover the opening 2/3 leaving 1/3 open for slow evaporation. Fill the cavity block every few hours for a minimum of 3 hours and no more than 1day with Solution II.

Solution II:

95 parts ethanol (96%)

5 parts glycerol

4. The next day the nematodes can then be permanently mounted on slides for identification and morphological studies.

5. To recover the DNA from nematodes permanently mounted remove the cover slip carefully with a straight razor. Remove the nematodes and wash with DI water by passing each nematode through a series of 3 dishes containing the DI water to remove the glycerol.

Results from Permanent Mounts

PCR of mounted nematodes:

Morphological results of nematodes removed from permanent mounts:

5M4H5 is from the UCR Botanic Gardens and was taken from a Rose plant.