Solutions used in Organelle Extraction


Introduction

Grinding Solution: is used for grinding cells before we extract cell organelles and lysis from cell fraction.
Suspension solution: is used to stabilize solution and also protect against any changes in test tube.
PBS: Phosphate-buffered saline is an organic buffer using for dilutions and when we need to shake test tube having biological substances , because it is isotonic and not toxic for many kinds of cells. Also it can be used with EDTA, by this way it can prevent cells attach to each other and container solution. 3
EDTA: can protect against heavy metal in solutions for samples and deactivate metal-dependent enzymes .4
TritonX: is a compound that is used to reduce surface tension of solutions while processes being detected to a specific protein especially in 0.1% concentration with PBS at biological experiments.5
Tris: Tris(hydroxymethyl)aminomethane is an organic compound that is used as solution buffer, and raise permeability of membrane.6
MgCl2 :an very soluble inorganic compound that has a role as cofactor for many enzymes and also it is used for PCR reaction with DNA polymerase as a component of solution.7
NaCl: is used for osmosis, having isotonic environment for cell culture. 8

Material and Methods:
We firstly calculated how much mass we need sucrose in gram for 4M, 2M, 0.25M by using the formula( m=MW*V*M) and we measured them on balance and then we placed them into bakers. Then we added water for each baker including sucrose until it is 50 ml. We used heater to get prepare solution faster. We placed solutions into 50 ml falcons( 4M and 2M solutions), and 15 ml falcon( 0.25M solution).
After sucrose solutions, for NaCl solutions(10M and 0.035M) , we calculated masses we need to prepare solutions. We measured them on balance. For 0.035 M NaCl , we added water into 50 ml falcons and for 10 M NaCl, we added water to 1 ml into eppendorf.
At PBS preparation, we got 5 ml PBS and we diluted 45 ml water to have 1*PBS from 10*PBS. Then we added 5µl TritonX into PBS solution to have 0.01% TirotonX in 1*PBS according to calculations.
For 0,1 M KH2PO4/ K2HPO4 buffer in 20ml, 0.2 M KH2PO4 in 10 ml and 0.2 M KH2PO4 in 10ml solutions were calculated their mass to make solutions and then we got 6.85 ml from KH2PO4 solution and Secondly we got 3.15 ml from K2HPO4 solution. Lastly, the solutions were mixed to have buffer.
Grinding solution, we made solutions according to calculations ( sodium pyrophosphate, MgCl2, Ascorbic acid, HCl). Then we mixed them except HCl to adjust 6.5 pH and then we added sorbitol until we have 20 ml grinding solution.
We also used same process for suspension solution. We mixed the components of suspension solution (Sorbitol, EDTA, MgCl2, HEPES, NaOH). The solutions were mixed except NaOH. Finally we used NaOH to adjust 7.6 pH.
Results:
For 2M sucrose solution, 50 ml:
m=MW*V*M, m= 342 g/mole*0.05L*2 mole/L=34.2 g
For 4M sucrose solution ,50 ml:
m= 342 g/mole*0.05L*4 mole/L=64.4 g
For 10M NaCl, 1 ml:
m= 58,44 g/mole*0.001 L*10mole/L= 0.5844 g
For 0.25M sucrose solution, 15 ml:
m= 342 g/mole*0.015ml*0.25mole/L=1,2825 g
For 0.035M NaCl solution, 30ml:
m=58.44 g/mole*0.030L*0.035mole/L=0,061362 g
For 1*PBS, 50ml:
C1*V1=C2*V2, 10*PBS=1*50ml, PBS=5ml
For 5*10-7 M DCMU, 1.5 ml:
m=233.09 g/mole*0.0015ml*5*10-7mole/L=0.0007 g
For 0,1 M KH2PO4/ KH2PO4 buffer, 20ml:
0.2 M KH2PO4 , 10 ml, m=174,18 g/mole*0.01L*0.2mole/L=0.35 g
0.2 M KH2PO4 , 10 ml, m=136.09 g/mole*0.01L*0.2mole/L=0.27 g
For 10-4 M DCPIP, 10ml:
m=268.09 g/mole*0.001L*10-4 mole/L=26.809 g
For %0.1 TritonX in 1*PBS:
0.1/100= X*5 ml( PBS) , X=5/1000 ml TritonX, X=5 µl
For Grinding solution, 20 ml:
0.33 M Sorbitol:
m=182 g/mole*0.02L*0.33 mole/L=1.2 g
10mM(0.01 M) Sodium pyrophosphate(Na4P2O7):
m=266 g/mole*0.02L*0,01 mole/L=0.0532 g
4mM(0.004 M) MgCl2:
m=95 g/mole*0.02L*0.004 mole/L=0.0076 g
2mM(0.002 M) Ascorbic acid:
m=176 g/mole*0.02L*0.02 mole/L=0.00704 g
HCl to adjust pH to 6.5
Suspension solution, 30 ml:
0.33 M Sorbitol:
m=182 g/mole*0.03L*0.33 mole/L=1.8 g
2mM(0.002M) EDTA:
m=292 g/mole*0.03L*0.002 mole/L=0.0175 g
1mM(0.001M) MgCl2:
m=95 g/mole*0.03L*0.001 mole/L=0.00285 g
50mM(0.05M) HEPES:
m=238 g/mole*0.03L*0.05 mole/L=0,357 g
NaOH to adjust pH to 7.6

Discussion:
MSDS(Material Safety Data Sheet) is an important guide that includes introductions about hazards, use of chemicals for human health and safety.9
The common parts of a chemicals in MSDS are hazards identification, information on ingredients, storage of chemical and handling ,physical and chemical properties, reactivity of chemical.10 We are using them by paying attention and following introductions. For example, we used PBS in this lab and for MSDS, firstly we used gloves against slashing any drops onto us and we used experimental equipment carefully by paying all our attention, then we transported it carefully to use for preparing solution after we closed tube firmly. Finally we cleaned all equipment and area where we used the chemical.
Some of common solvents are water, isopropanol, acetic acid, chloroform, formic acid, acetone. 11We use PBS to dissolve TritonX for having lower surface tension and also PBS is a soluble nontoxic and isotonic solvent. We could use another solvents to dissolve TritonX without PBS. It could be water, or TBS(Tris-buffered saline). TBS12 has same properties with PBS.
References:

[1]  Ji, Hong (2010-08-01). “Lysis of Cultured Cells for Immunoprecipitation”. Cold Spring Harbor Protocols. 2010 (8): pdb.prot5466. doi:10.1101/pdb.prot5466. ISSN 1940-3402. PMID 20679375
[2] Chemistry: Matter and Its Changes, 4th Ed. by Brady, Senese, ISBN 0-471-21517-1
[3]  Medicago AB, (2010) Phosphate buffered saline specification sheet
[4] Dominguez, K; Ward, WS (December 2009). “A novel nuclease activity that is activated by Ca2+ chelated to EGTA”. Systems Biology in Reproductive Medicine. 55 (5–6): 193–99.doi:10.3109/19396360903234052
[5] “Triton X-100″. exactantigen.com. Retrieved 2009-10-22.
[6] Irvin, R.T.; MacAlister, T.J.; Costerton, J.W. (1981). “Tris(hydroxymethyl)aminomethane Buffer Modification ofEscherichia coli Outer Membrane Permeability”. J. Bacteriol. 145 (3): 1397–1403
[7] Bales, S. E.; Hudnall, P. M.; Burns, T. P.; Poindexter, G. S. “Highly Reactive Magnesium for the Preparation of Grignard Reagents: 1-Norbornane Acid”, Organic Syntheses, Collected Volume 6, p. 845 (1988).
[8]  Lincoln, S. F.; Richens, D. T. and Sykes, A. G. (2003) “Metal Aqua Ions” Comprehensive Coordination Chemistry II Volume 1, pp. 515–555. doi:10.1016/B0-08-043748-6/01055-0
[9]  Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS)
[10]http://eurlex.europa.eu/Notice.do?val=516832:cs&lang=pl&list=643062:cs,522140:cs,521353:cs,516832:cs,&pos=4&page=1&nbl=4&pgs=10&hwords=&checktexte=checkbox&visu=#texte Archived October 29, 2013, at the Wayback Machine.
[11]  Tinoco, Ignacio; Sauer, Kenneth and Wang, James C. (2002)Physical Chemistry Prentice Hall p. 134 ISBN 0-13-026607-8
[12] Medicago AB, (2010) Phosphate buffered saline specification sheet

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