User:Nadiezda FernandezOropeza/Notebook/Notebook/2010/10/12
From OpenWetWare
(→Preparation of 10x PEM buffer solution) 
m (→Summary) 

Line 55:  Line 55:  
===Summary===  ===Summary===  
* To prepare the ~10N NaOH solution:  * To prepare the ~10N NaOH solution:  
  <center><math>#g NaOH*\frac{1 mole NaOH}{40 g NaOH}*\frac{1000mL H_{2}O}{10 moles NaOH}= (#) mL H_{2}O</math></center>  +  <center><math>(#) g NaOH* \frac{1 mole NaOH}{40 g NaOH}*\frac{1000mL H_{2}O}{10 moles NaOH}= (#) mL H_{2}O</math></center> 
*To prepare a 25 mL solution:  *To prepare a 25 mL solution:  
  <center><math>25 mL sol. * \frac{800*10^{3}moles PIPES}{1000 mL sol}*\frac{302.37 g PIPES}{1 mole PIPES}= 6.0474 g PIPES</math></center>  +  <center><math>25 mL sol. * \frac{800*10^{3}moles PIPES}{1000 mL sol}*\frac{302.37 g PIPES}{1 mole PIPES}= '''6.0474 g PIPES'''</math></center> 
  <center><math>25 mL sol. * \frac{10*10^{3} moles of EGTA}{1000 mL sol.}*\frac{380.35 g EGTA}{1 mole EGTA} = 0.950875 g EGTA</math></center>  +  <center><math>25 mL sol. * \frac{10*10^{3} moles of EGTA}{1000 mL sol.}*\frac{380.35 g EGTA}{1 mole EGTA} = '''0.950875 g EGTA'''</math></center> 
  <center><math>25 mL sol. * \frac{1.25 moles NaOH}{1000 mL sol}*\frac{40 g NaOH}{1 mole NaOH}= 1.25 g  +  <center><math>25 mL sol. * \frac{1.25 moles NaOH}{1000 mL sol}*\frac{40 g NaOH}{1 mole NaOH}= '''1.25 g NaOH'''</math></center> 
  <center><math>25 mL sol. * \frac{10*10^{3}moles MgCl_{2}}{1000 mL sol}*\frac{1000 mL sol. MgCl_{2}}{1 mole MgCl_{2}}= 0.25 mL sol. MgCl_{2}</math></center>  +  <center><math>25 mL sol. * \frac{10*10^{3}moles MgCl_{2}}{1000 mL sol}*\frac{1000 mL sol. MgCl_{2}}{1 mole MgCl_{2}}= '''0.25 mL sol. MgCl_{2}'''</math></center> 
Add PIPES and EGTA in a 50 mL tube, and then add a small amount of distilled water and a pellet of NaOH (previously weighted). Centrifuge, and add the MgCl2. Measure the pH and proceed with titration with NaOH (~10N) until pH 6.9 is obtained.  Add PIPES and EGTA in a 50 mL tube, and then add a small amount of distilled water and a pellet of NaOH (previously weighted). Centrifuge, and add the MgCl2. Measure the pH and proceed with titration with NaOH (~10N) until pH 6.9 is obtained. 
Revision as of 19:12, 16 February 2012
Project name  Main project page Previous entry Next entry  
Preparation of 10x PEM buffer solution
The 10x PEM buffer solution is simple the PEM solution 10 times more concentrated. PEM buffer has the following components: PIPES, EGTA, MgCl_{2}, NaOH and distilled water. To have a better description of each of these chemicals see: PEM buffer The recipe for 10x PEM is: 800 mM PIPES 10 mM EGTA 10 mM MgCl_{2} ~ 1.25M NaOH pH 6.89
This means that this is the concentration of each of the components in solution. PIPES and EGTA are in the form of powder, so the best way to get the needed amount of them is to first gently and slowly pour the chemical into the weighing boat on the scale. Once you reach a close enough quantity to the one you need stop and pour a little bit more of the chemical on a separate weighing boat and from there, with the help of a spatula Oddly enough, they are called spoonulas, add the necessary quantities little by little. After placing the chemicals in the test tubes, you will realize that not all the chemical was poured down. Figure out the difference, and measure that remaining quantity in a wax paper. Complete the process. Due to the hygroscopic nature of NaOH, the collection of this chemical needs to be fast. Originally, they are in form of pellets, so it is hard to get the exact amount needed, but just try to get as close as you can. Since they are pellets, the collection needs to be done with tweezers. Once both acids are on the tube, add small amounts of distilled water, always leaving enough room for the remaining chemicals. Vortex the tube, then you will observe a milklike color on the tube. This means that the acids did not dissolved completely. To help the acids dissolve completely and to reach the desired pH, we add the pellets of NaOH. Vortex the tube and the solutions should be crystal clear. After, add the necessary amount of MgCl_{2} to the solution with the micro pipettes. To see how to properly use the pipettes check the following link:[Micro Pipettes]
Once this is ready add some volume of distilled water, leaving enough room for titration. This is when the process of obtaining the desired pH begins. To see how to properly use a pH meter see the following link: pH meter Measure the pH of the solution the way it is. You will observe that the pH will be below the expected value 6.89. Thus, you need to add small amounts of a base, in this case NaOH (a solution ~10 N would be best), until the solution reaches a pH of 6.89± 1. Once, that pH is achieved, complete the missing value with distilled water and measure the pH again. You will notice something interesting: the pH of the solution dropped by some hundredths and this is due to the slight acidic nature of the water we work with. Finally, prepare 2 ml aliquots of the solution. Make sure to label correctly each single one of them, and store them in the freezer. Summary
Add PIPES and EGTA in a 50 mL tube, and then add a small amount of distilled water and a pellet of NaOH (previously weighted). Centrifuge, and add the MgCl2. Measure the pH and proceed with titration with NaOH (~10N) until pH 6.9 is obtained. Prepare 2 ml aliquots of the solution and store in the freezer.
