Contents

Introduction

The focus of this work is describing and testing a known low-cost protocol for DNA extraction from S. cerevisiae, and then trying to apply it for E. coli, while at the same time proposing a novel natural and cheap reactive to accomplish the chelating need that is normally carried out by EDTA.

Main proposal: Garlic and grape juice in order to replace EDTA in Miniprep protocols.

Several studies have shown that garlic as well as malic acid (mainly found in grapes and apples) have strong chelating capacities, and several therapies for metal detoxification using these components have been created (Flora & Pachauri, 2010; Sears, 2013).

Garlics have sulphur-rich molecules, and studies have revealed how these compounds have high affinity for metals such as lead and cadmium. On the other hand, malic acid can sequester many metals such as Pb, Cd, Zn and Cu (Ding, Song, Feng, & Guo, 2014). We deduce that since EDTA can sequester both toxic and nontoxic metals (cofactors), then garlic and malic acid have also high probabilities of being able to accomplish this feat.

Testing and results

The protocol that was used is the standard protocol for DNA extraction by miniprep. Only, some reagents were changed, as reported in the following table. The results for DNA purity and quantity obtained are also reported below.

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Test number Description Reagents Results: DNA concentration and purity (Mean of both starter cultures)(Appendix A)
1 (S. cerevisiae)
(Proof of concept)
  • Solution I Lemon and salt
  • Solution II Detergent
  • Solution III Salt
  • DNA concentration = 4101.43 ng/L
  • DNA/Protein ratio= 1.75
  • DNA/Salt ratio = 1.5
2 (E.coli)
Control (normal)
  • Solution I. (50 mM TRIS pH 8.0, 10 mM EDTA)
  • Solution II (200 mM NaOH, 1% w/v SDS)
  • Solution III (3 M potassium acetate, pH 5.5)
  • DNA concentration = 1179.89 ng/L
  • DNA/Protein ratio= 1.965
  • DNA/Salt ratio = 2.46
3 (E.coli)
Normal protocol using garlic and grape juice instead of EDTA
  • Protocol 2
  • Solution I. (50 mM TRIS pH 8.0, concentrated garlic and grape juice)
  • Solution II (200 mM NaOH, 1% w/v SDS)
  • Solution III (3 M potassium acetate)
  • DNA concentration = 1053.09 ng/L
  • DNA/Protein ratio= 1.94
  • DNA/Salt ratio = 2.345
4 (E.coli)
Novel modified protocol
  • Protocol 2
  • Solution I. (25 mM TRIS pH 8.0*, concentrated garlic and grape juice)
  • Solution II (Axion Detergent)
  • Solution III (3 M NaCl)
  • DNA concentration = 636.6 ng/L
  • DNA/Protein ratio= 1.54
  • DNA/Salt ratio = 1.01

Note: Solution I = Buffer and Chelator, Solution II= Lysis, Solution III= Protein precipitation

Cost comparison (Ethanol is used in all tests, so it is omitted) (It is assumed that approximately equal quantities of each analogue reagent are used)

<col style="width:20%"> <col style="width:20%"> <col style="width:15%"> <col style="width:15%"> <col style="width:15%"> <col style="width:15%">
Article (1kg or 1L) Price (American Dollars) Test 1 Test 2 Test 3 Test 4
TRIS
109.20
*
*
*/2
EDTA
74.25
*
SDS
113.01
*
*
NaOH
2.5
*
*
Lemon
1
*
Garlic
6.5
*
*
Grape juice (Assuming 1kg grape = 1L grape juice)
4
*
*
*
Table Salt (NaCl)
0.6
*
*
Axion Detergent
1
*
*
TOTAL per kg/L
-
6.6 dollars
298.96 dollars
235.21 dollars
66.7 dollars

Conclusion

With the use of protocol from test 1, 292.36 dollars are saved. From test 3, 63.5 dollars can be saved from each kg or L used. Using solutions from test 4, 232.26 dollars are saved. It is easy to see that these alternatives have to be considered and further evaluated to incorporate them as a whole or parts of it as routine iGEM procedures, to contribute to making protocols more accessible and feasible for low resources laboratories.


References

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  1. Bio-Rad Laboratories Inc. (2014). Bio-Rad. Retrieved from Bio-Rad: www.bio-rad.com

  2. Ding, Y. Z., Song, Z. G., Feng, R. W., & Guo, J. K. (2014). Interaction of organic acids and pH on multi-heavy metal extraction from alkaline and acid mine soils. International Journal of Environmental Science and Technology, 11(1), 33–42. doi:10.1007/s13762-013-0433-7

  3. <p> Flora, S. J. S., & Pachauri, V. (2010). Chelation in metal intoxication. International Journal of Environmental Research and Public Health, 7(7), 2745–88. doi:10.3390/ijerph7072745

  4. Marshall University. (2012). DNA isolation methods. Retrieved from Marshall University: http://science.marshall.edu/murraye/links%20for%20students/samantha%20qiagin%20method.pdf

  5. Sears, M. E. (2013). Chelation: harnessing and enhancing heavy metal detoxification--a review. TheScientificWorldJournal, 2013, 219840. doi:10.1155/2013/219840

  6. Sigma-Aldrich. (2014). Products. Retrieved from Sigma-Aldrich: http://www.sigmaaldrich.com/

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