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DMSO/EDTA/NaCl protocol - dmso bladder instillation protocol

DESS (DMSO/ EDTA/ NACl) protocol

0.25M disodium EDTA pH 8.0
20% DMSO (Dimethyl Sulfoxide)
NaCl saturated

**The most difficult and time-consuming part of making DESS solution is waiting for the EDTA to dissolve therefore, to making it easier making a large volume of EDTA at double the molarity (0.5M EDTA) that can used at a moments notice is best. Below are the directions for making large volumes of DESS by making a stock solution of 0.5M EDTA and how to make small volumes of DESS the original/difficult way by making 0.25M EDTA. Both directions can be easily adapted to make either small or large volumes of EDTA. NOTE: Be patient when making any EDTA solution because it will not even begin to dissolve until it reaches a pH of ~ 7.
**See the FAQ section below for answers about the formation of crystals at the bottom of the solution. If you still have question email me (Melissa Yoder) at melissay@ucr.edu and I will do my best to answer your questions.

New easier way to make DESS:
First make a stock solution of 0.5M EDTA, if the sock solution is already present then proceed to the section below that says ‘2L DESS.’

2L 0.5M disodium EDTA
|Disodium EDTA FW 374.24 |372.24g |
|5M NaOH |To pH EDTA to 8.0 |
|Deionized water |Bring final volume to 2L |

1. Measure out 372.24g of disodium EDTA, add 500mL deionized water to the EDTA, and enough 5M NaOH to pH the solution to 8.0 (this can be as much as 500mL in some cases). The EDTA will start to dissolve around a pH of 7.0, which may take several hours, so be patient.
**Make sure to use disodium EDTA salt otherwise more NaOH is needed to pH the EDTA.
2. Bring the final volume to 2L with deionized water.

|0.5M disodium EDTA |1L |
|Dimethyl Sulfoxide (DMSO) |400mL |
|Deionized water |600mL |
|NaCl |Enough to saturate the solution ~300g |

1. Measure out and mix the first three chemicals.
2. Add enough NaCl to saturate the solution (roughly 300-400g, this may vary depending on several factors including ambient temperature, etc so is best to add 300 grams and if everything dissolves then add more salt until it seems like it just isn’t dissolving anymore) and dissolve. This may take several minutes to hours, so be patient.

The original way to make DESS:
To make small volumes of DESS you can either adapt the directions above or use the directions below by making 0.25M EDTA for the volume of DESS needed.
250mL DESS
|disodium EDTA FW 374.24 |23.265 g |
|1M NaOH |To pH EDTA to 8.0 |
|Dimethyl Sulfoxide (DMSO) |50 mL |
|NaCl |Enough to saturate the solution |
|Deionized water |Enough to bring the final volume up to |
| |250mL |

1. Measure out 23.265g of disodium EDTA 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 use disodium EDTA 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 8.0. 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 by volume, 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, heating will help dissolve the salt. Pour the solution into a bottle leaving most of the salt crystals in the beaker. (The next day some salt may have settled or precipitated at the bottom of the bottle, do not worry about trying to dissolve this salt.)

Frequently asked questions:
1) There are crystals at the bottom of my solution, is this normal?
Yes. Because the solution is saturated with NaCl it is normal for the salt to precipitate out of the solution once the liquid settles. If there are an excessive amount of crystals on the bottom of the container it is best to move the solution into a new container leaving the crystals in the other bottle. The other thing is to make sure that the container is sealed tightly to reduce the evaporation of the water in the solution, which will also result in precipitation of the NaCl. When you not using the solution make sure that it is placed in an airtight (or as close to airtight as possible) container.
The crystals may also appear at bottom of preserved samples and if it is a bother the sample can be rinsed in water to remove the salt and put back in DESS. If you are going to use a sample preserved in DESS the next day and don’t want to risk losing worms by moving the sample in and out of a vial the thing to do is covering the dish and put them in a fridge (around 4-10C should do). This should prevent the liquid from evaporating quickly leaving only salt crystals behind.
2) How do I ship samples to my colleagues in another state or country?
a) Contact your local health and safety organization for an official letter of shipment, which should include the chemical MSDS (from a certified chemist) to be included in the package. Be sure to include a statement from the shipper about the content of the box and general purpose of the material
b) Seal all containers tightly by wrapping each one in parafilm to prevent leaking. Put sealed samples inside a plastic bag; place that plastic bag in another plastic bag with aluminosilicate (or an alternative absorbent), and seal. Place the bag in a box with additional packing material, seal, and send.

How it works:
DESS works by inactivating nucleases that will degrade DNA upon cell death and preventing DNA denaturation at room temperature because of the high concentration of salt. Dimethyl Sulfoxide works by permeating cells and takes low molecular weight chemicals with it like EDTA and NaCl. Once inside the cell the EDTA chelates divalent cations, which are needed for nucleases to activate and degrade DNA. The Na+ ions from the NaCl will surround the negatively charged DNA phosphate backbone preventing water molecules from coming in contact with the hydrogen bonds of the base pairs and denaturing the double strands.

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.

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.

Yoder, M., I. Tandingan De Ley, I. Wm. King, M. Mundo-Ocampo, J. Mann, M. Blaxter, L. Poiras, and P. De Ley. 2006. DESS: A versatile solution for preserving morphology and extractable DNA of nematodes. Nematology, 8(3): 367-376.

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 DESS solution to the side of the sieve where the nematodes are concentrated. Tilt the sieve with DESS over a vial and rinse the mesh with DESS 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 DESS solution is that they shrivel slightly and return to their original form after being in the solution about 15 minutes.

Extracting samples from DESS

1. Pick the nematodes you want from the DESS solution and place in a dish with water for a few minutes to remove any salt or DESS that might be attached. If the DESS is not thoroughly washed off the dimethyl sulfoxide and EDTA will inhibit the PCR reaction.

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

A) B)
[pic] [pic]

C) D)
[pic] [pic]

E) F)
[pic] [pic]

G) H)
[pic] [pic]

Good Morphological Preservation
A) 5M7G5 from Kern Co. sample that was placed in DESS 5/13/05 and capture 7/7/05.
B) 3M7G5 is from Saharna, Moldova was placed in DESS 6/28/05 and was captured 7/7/05.
C) 4W5G5 is from the Black Sea was placed in DESS 6/25/05 and was captured 7/5/05.
D) 8M2F5 is from the UCR Botanic Gardens was placed in DESS 5/13/05 and was captured 6/2/05.
Bad Morphological Preservation (All of these nematodes came from freshwater sediment, which is part of the reason the morphological preservation is not very good.)
E) 3M5G5 from Pruit River, Moldova in DESS 6/28/05 and captured 7/5/05
F) 4M5G5 from Pruit River, Moldova in DESS 6/28/05 and captured 7/5/05
G) 5M5G5 from Pruit River, Moldova in DESS 6/28/05 and captured 7/5/05
H) 6M5G5 from Pruit River, Moldova in DESS 6/28/05 and captured 7/5/05

Making Permanent Mounts

1. Wash the sample in the DESS solution on a #500 mesh sieve with DI water until all the DESS solution 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

1. Nematode from UCRBG Rosa cv 5/13.05 sample were transferred to glycerin and 100 were mounted on permanent slides. One slide was broken open and nematodes were washed before VCE+PCR.

Morphological results of nematodes removed from permanent mounts:
[pic] [pic]
5M4H5 is from the UCR Botanic Gardens and was taken from a Rose plant.

Extracting Nematodes from DESS Preserved Soil with Ludox.
1. DESS solution was directly added to soil and mixed thoroughly. Samples were stored at room temperature for up to three weeks.
2. Make Ludox at a 2:3 dilution (2 of Ludox to 3 of water) for use later. Ludox is not dangerous in liquid form but can be if allowed to dry. Clean up any spills immediately with water and NaOH if necessary. All equipment should be rinsed in NaOH after being exposed to Ludox.
3. Place preserved soil in a stainless steel bucket and fill half full with water. Mix the soil thoroughly breaking up any large particles. Pour immediately over a #80 sieve (sieve number will vary depending on the soil type) retaining the water in a second bucket and rinse the sieve well.
4. Mix the water in the second bucket and allow to sit for 30 seconds, pour over a #500 sieve slowly. Rinse the sieve by tilting at a 45-degree angle concentrating all of the soil to one corner and place in a labeled beaker. Add more water to the first bucket and start over again.
5. Repeat steps 3 and 4 at least two more times for all samples.
6.Pour reserved soil into 50ml centrifuge tubes without filling the tube more than 10ml full of soil. Multiple centrifuge tubes may be needed for a single sample.
7. Fill each centrifuge tube up to 50ml with water. Place into a swinging centrifuge and balance if needed. Spin at 2500rpm for 5 minutes. Remove each sample, take the supernatant off with a pipette and reserve it in a labeled beaker.
8. Add diluted Ludox up to 50ml for each centrifuge tube and mix thoroughly making sure to disturbing the soil (using a spatula helps). Place in the centrifuge and spin at 2500rpm for 15 minutes.
9. Remove the supernatant with a pipette and add it to the beaker used in step 7.
10. Repeat steps 8 and 9 at least 2 more times for each sample.
11. Take the reserved supernatant and pour through a #500 sieve. Rinse well to remove any excess Ludox. Rinse with DESS solution and place in a labeled vial for VCE+PCR when needed.

Results from Ludox Extraction

1. Three different samples were preserved in DESS. These were 1) Sediment from the Salton Sea collected 8/10/05, 2) Soil from a patch of flowers below our lab building collected 8/15/05, and 3) Soil from BBQ Hill at UCR collected 7/25/05. 2. All three were extracted with Ludox and six nematodes from each sample were used for VCE+PCR.
PCR Results:

Morphological results:
A) B)
[pic] [pic]
A) 5M17H5 is from the Salton Sea sample.
B) 8M17H5 is from the soil collected below our lab.
C)* 13M17H5 is from the BBQ Hill sample.
*This sample was not immediately mixed and many of the specimens were not preserved very well.

Any questions can be sent to Melissa Yoder at melissay@ucr.edu

What is the DMSO/EDTA/NaCl protocol? DESS (DMSO/ EDTA/ NACl) protocol Recipe: 0.25M disodium EDTA pH 8.0 20% DMSO (Dimethyl Sulfoxide) NaCl saturated