Protocol for the preparation of Nucleic Acid Preservation (NAP) Buffer
######
Full reference:
Camacho‐Sanchez, M., Burraco, P., Gomez‐Mestre, I., & Leonard, J. A. (2013). Preservation of RNA and DNA from mammal samples under field conditions. Molecular Ecology Resources, 13(4), 663-673. [link]
######
Materials:
EDTA disodium salt dihydrate (CAS # 6381-92-6)
Sodium citrate trisodium salt dihydrate (CAS # 6132-04-3)
Ammonium sulfate (CAS # 7783-20-2)
Ultra-purified, molecular grade water
H2SO4 or HCl to adjust the pH (use appropriate solution. Read MSDS)
Recipe (thanks to Kejal Dodhia for updating the recipe):
Equipment:
Bottle or flask
Scale
Weigh boat or paper
Magnetic stirrer with heating plate
Stirring rod
PH reader
To make NAP buffer:
1 Combine 7.44 g of EDTA, 7.35 g of sodium citrate trisodium salt dihydrate, and 700 g of ammonium sulfate in 1 L of water in bottle or flask. Stir on low to moderate heat until the ammonium sulfate dissolves completely, which usually takes hours.
**[[WARNING: since the volume of the solution increases when adding ammonium sulfate up to about 1.4L, your calculations on molarity won't match those described in the publication. So please, stick to the recipe in point 1 or quantities in equivalent proportions.]]**
#recommendation: this large amount of ammonium sulfate is hard to dissolve in water. If the RPMs of the magnetic stirrer are too high, then the rod will rotate with such a high frequency that in will produce very little turbulence and the ammonium sulfate will not dissolve completely. My recommendation is play with the adjustment of the RPMs to maximize the oscillations of the rod to create a maximum of turbulences. Under these optimal conditions the salts should dissolve in less than 1h.
2 Cool to room temperature, then adjust pH to 5.2.
3 Store at room temperature or keep refrigerated until aliquoted.
4 Aliquot 1.5 mL of buffer into 2 mL tubes for preservation of up to 150 mg of sliced tissue.
####################################
REFERENCES (updated on August, 30th 2019):
NAP buffer has been successfully used to preserve nucleic acids in:
1. DNA and RNA from muscle, liver and ear in small mammals:
- https://doi.org/10.1111/1755-0998.12108
- https://doi.org/10.1111/ddi.12761
- https://doi.org/10.1093/jmammal/gyx081
2. RNA from brain in birds:
- https://doi.org/10.1016/j.ygcen.2015.05.016
3. RNA in fungi:
- https://doi.org/10.1186/s12864-016-2952-3
- https://doi.org/10.1016/j.mimet.2016.05.021
4. Microbiome (DNA) for metagenomics:
- https://doi.org/10.3389/fmicb.2017.00102
5. DNA in fecal samples:
- https://repositories.lib.utexas.edu/bitstream/handle/2152/38629/ABONDANO-THESIS-2014.pdf?sequence=1
6. RNA from intestine, liver, spleen, heart and skin, in amphibians:
- 10.7717/peerj.3702
7. Insects:
- https://www.researchgate.net/profile/Nathan_Lord/publication/303540962_The_Effects_of_Non-Ideal_Temperature_Regimes_on_RNA_Quality_from_Samples_Stored_in_RNAlater-like_Buffer_An_Attempt_to_Replicate_Field_Conditions/links/574714db08ae2301b0b7fdbb.pdf
8. RNA from testis in octopus
- https://doi.org/10.3389/fphys.2018.01920
9. RNA in fish:
- https://doi.org/10.1016/j.fsi.2020.01.054
- 10.1111/are.14455
10. Plants
- doi:10.1002/evl3.142
######
Full reference:
Camacho‐Sanchez, M., Burraco, P., Gomez‐Mestre, I., & Leonard, J. A. (2013). Preservation of RNA and DNA from mammal samples under field conditions. Molecular Ecology Resources, 13(4), 663-673. [link]
######
Materials:
EDTA disodium salt dihydrate (CAS # 6381-92-6)
Sodium citrate trisodium salt dihydrate (CAS # 6132-04-3)
Ammonium sulfate (CAS # 7783-20-2)
Ultra-purified, molecular grade water
H2SO4 or HCl to adjust the pH (use appropriate solution. Read MSDS)
Recipe (thanks to Kejal Dodhia for updating the recipe):
- 1L of water
- 0.744g % w/v EDTA disodium salt dihydrate
- 0.735 % w/v sodium citrate trisodium salt dehydrate
- 70 % w/v ammonium sulfate
- Adjust pH to 5.2 with H2SO4 or HCl
Equipment:
Bottle or flask
Scale
Weigh boat or paper
Magnetic stirrer with heating plate
Stirring rod
PH reader
To make NAP buffer:
1 Combine 7.44 g of EDTA, 7.35 g of sodium citrate trisodium salt dihydrate, and 700 g of ammonium sulfate in 1 L of water in bottle or flask. Stir on low to moderate heat until the ammonium sulfate dissolves completely, which usually takes hours.
**[[WARNING: since the volume of the solution increases when adding ammonium sulfate up to about 1.4L, your calculations on molarity won't match those described in the publication. So please, stick to the recipe in point 1 or quantities in equivalent proportions.]]**
#recommendation: this large amount of ammonium sulfate is hard to dissolve in water. If the RPMs of the magnetic stirrer are too high, then the rod will rotate with such a high frequency that in will produce very little turbulence and the ammonium sulfate will not dissolve completely. My recommendation is play with the adjustment of the RPMs to maximize the oscillations of the rod to create a maximum of turbulences. Under these optimal conditions the salts should dissolve in less than 1h.
2 Cool to room temperature, then adjust pH to 5.2.
3 Store at room temperature or keep refrigerated until aliquoted.
4 Aliquot 1.5 mL of buffer into 2 mL tubes for preservation of up to 150 mg of sliced tissue.
####################################
REFERENCES (updated on August, 30th 2019):
NAP buffer has been successfully used to preserve nucleic acids in:
1. DNA and RNA from muscle, liver and ear in small mammals:
- https://doi.org/10.1111/1755-0998.12108
- https://doi.org/10.1111/ddi.12761
- https://doi.org/10.1093/jmammal/gyx081
2. RNA from brain in birds:
- https://doi.org/10.1016/j.ygcen.2015.05.016
3. RNA in fungi:
- https://doi.org/10.1186/s12864-016-2952-3
- https://doi.org/10.1016/j.mimet.2016.05.021
4. Microbiome (DNA) for metagenomics:
- https://doi.org/10.3389/fmicb.2017.00102
5. DNA in fecal samples:
- https://repositories.lib.utexas.edu/bitstream/handle/2152/38629/ABONDANO-THESIS-2014.pdf?sequence=1
6. RNA from intestine, liver, spleen, heart and skin, in amphibians:
- 10.7717/peerj.3702
7. Insects:
- https://www.researchgate.net/profile/Nathan_Lord/publication/303540962_The_Effects_of_Non-Ideal_Temperature_Regimes_on_RNA_Quality_from_Samples_Stored_in_RNAlater-like_Buffer_An_Attempt_to_Replicate_Field_Conditions/links/574714db08ae2301b0b7fdbb.pdf
8. RNA from testis in octopus
- https://doi.org/10.3389/fphys.2018.01920
9. RNA in fish:
- https://doi.org/10.1016/j.fsi.2020.01.054
- 10.1111/are.14455
10. Plants
- doi:10.1002/evl3.142