# Goal/Experiment:
Preparation of iron chloride resuspension buffer using disodium EDTA dihydrate and magnesium chloride in Tris buffer.
# 0.1M EDTA-0.2M MgCl2-0.2M Ascorbate Buffer
## Abstract
Preparation of iron chloride resuspension buffer using disodium EDTA dihydrate and magnesium chloride in Tris buffer.
Citation: Seth John, Bonnie Poulos, Christine Schirmer 0.1M EDTA-0.2M MgCl2-0.2M Ascorbate Buffer. protocols.io dx.doi.org/10.17504/protocols.io.c2yyfv
Published: 20 Jul 2015
## Guidelines
### Recipe as developed by Seth:
| Reagent | Formula Weight | Amount | Final Concentration |
|----------------------------|----------------|---------|---------------------|
| Tris-base | FW=121.14 | 1.51g | 0.125M |
| Na₂-EDTA dihydrate | FW= 372.24 | 3.72g | 0.1M |
| MgCl₂ hexahydrate | FW=203.3 | 4.07g | 0.2M |
| Ascorbic Acid | FW=176.12 | 3.52g | 0.2M |
| 5N NaOH | | ~4.0ml | to pH 6.5 final |
| MilliQ H₂O | | to 100ml| |
1. **Tris-base**: A common buffering agent.
2. **Na₂-EDTA dihydrate**: Chelating agent to bind divalent cations.
3. **MgCl₂ hexahydrate**: Source of Mg²⁺ ions.
4. **Ascorbic Acid**: Reductant to improve virus infectivity.
5. **5N NaOH**: Used to adjust pH.
*Oxalic acid can be substituted for ascorbic acid to improve virus infectivity. Use oxalic acid dihydrate (FW=126.07) at 2.52g/100ml for 0.2M.*
### 2X Ascorbic Acid Buffer:
Keep the amount of Tris-base, water, and NaOH the same, but double the amount of EDTA, Mg and ascorbate. Check the pH and add NaOH or HCl to get final pH to 6.5. If increasing 2x, use 1 ml for every 2 mg Fe.
### Notes:
- The new formulation uses EDTA and MgCl₂.
- Ensure EDTA has a pH above 8.0 to dissolve.
- Ascorbic acid may come out of solution if the pH is very high (above 5.0).
### Recipe tested with diluted amounts of key reagents:
| Reagent | ½ Na₂-EDTA (1.86g/100ml) | ½ MgCl₂ (2.04g/100ml) | ½ Oxalic Acid-2H₂O (1.46g/100ml) |
|---------------------------|--------------------------|------------------------|-----------------------------------|
| Tris-base 1.51g/100ml | clear; pH 10.79 | clear; pH 10.82 | clear; pH 10.78 |
| Na₂-EDTA 3.72g/100ml | clear | clear | clear |
| MgCl₂·6H₂O 4.07g/100ml | clear; pH 7.68 | clear; pH 4.89 | clear; pH 4.59 |
| 5N NaOH | none; pH 7.68 | 1.25ml; pH 7.23 | 1.5ml; pH 7.51 |
| Oxalic acid·2H₂O 2.52g/100ml | white; pH 1.68 | cloudy; pH 3.02 | white; pH 3.30 |
- Final results showed best results with ½ MgCl₂, intermediate results with ½ Oxalic acid, and worst results with ½ Na₂-EDTA.
## Protocol
### 1x Buffer
#### Step 1.
Dissolve 1.51g Tris-base in 80ml Milli Q water.
#### Step 2.
Dissolve 3.72g Na₂-EDTA dihydrate into solution.
> **Note:** pH will be ~10.0
#### Step 3.
Once EDTA is in solution, dissolve 4.07g MgCl₂.
> **Note:** pH will drop to ~8.0
#### Step 4.
Add 3ml of NaOH.
> **Note:** This will drop the pH to ~4.5 and the solution will become cloudy indicating that the EDTA is coming out of solution.
#### Step 5.
Dissolve the reductant (3.52g of ascorbic acid or 2.52g of oxalic acid).
> **Note:** The pH will increase to ~8.3 and the solution will clear up.
#### Step 6.
Once the reductant is in solution, add the last 1ml of NaOH.
#### Step 7.
Check the pH using pH paper (the buffer should be at pH 6.0 - 6.5).
> **Note:** The solution may need some minor adjusting with NaOH or HCl to achieve a pH of 6.0, which is ideal for good recovery of viruses.
#### Step 8.
Check the volume and add MilliQ water for a total volume of 100ml.
#### Step 9.
Store the buffer in the dark (bottle wrapped in foil) and visually inspect prior to use. It should be clear without precipitates.
> **Note:** The buffer will start to change color after about 24 hours but it is okay to use if slightly discolored. Do not use after about 36 hours.
## Warnings
- EDTA needs a pH above 8.0 to dissolve and will come out of solution at pH below 5.0.
- Ascorbic acid may come out of solution if the pH is very high.
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