(Difference between revisions)
 Revision as of 19:12, 16 February 2012 (view source)m (→Summary)← Previous diff Current revision (19:12, 16 February 2012) (view source)m (→Summary) Line 55: Line 55: ===Summary=== ===Summary=== * To prepare the ~10N NaOH solution: * To prepare the ~10N NaOH solution: -
$(#) g NaOH* \frac{1 mole NaOH}{40 g NaOH}*\frac{1000mL H_{2}O}{10 moles NaOH}= (#) mL H_{2}O$
+
$g NaOH* \frac{1 mole NaOH}{40 g NaOH}*\frac{1000mL H_{2}O}{10 moles NaOH}= mL H_{2}O$
*To prepare a 25 mL solution: *To prepare a 25 mL solution:
$25 mL sol. * \frac{800*10^{-3}moles PIPES}{1000 mL sol}*\frac{302.37 g PIPES}{1 mole PIPES}= '''6.0474 g PIPES'''$
$25 mL sol. * \frac{800*10^{-3}moles PIPES}{1000 mL sol}*\frac{302.37 g PIPES}{1 mole PIPES}= '''6.0474 g PIPES'''$

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## Preparation of 10x PEM buffer solution

• Note for the preparation of the 10 x PEM buffer solutions

The 10x PEM buffer solution is simple the PEM solution 10 times more concentrated. PEM buffer has the following components: PIPES, EGTA, MgCl2, 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 MgCl2

~ 1.25M NaOH

pH 6.89

• Andy Maloney 14:01, 26 October 2010 (EDT): So you don't have to go into math mode to do subscripts or superscripts. You just have to type <sub></sub> for a subscript or <sup></sup> for a superscript.

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.

 Getting the missing amount of a chemical Getting the excess of a chemical

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 milk-like 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 MgCl2 to the solution with the micro pipettes. To see how to properly use the pipettes check the following link:[Micro Pipettes]

• Andy Maloney 14:01, 26 October 2010 (EDT): When you are linking an outside website in the wiki, you put single square brackets around the URL [http://www.interestingwebsite... Name]. With the space and the name you want to see in the markup. When you are linking to a page within the wiki, like the one above, you put double brackets around the link [[Koch_Lab:Protocols/How_to_use_a_pipette Micro Pipettes|Pipettors]] and you only add the stuff in the URL that is after the wiki. For instance, [[http://openwetware.org/wiki/...]] is not added to the link. If you want to name the wiki link to show up in the markup, you have to put the vertical line character | and then the name you want to see. Just look at the markup to see what I'm talking about.

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

• To prepare the ~10N NaOH solution:
$g NaOH* \frac{1 mole NaOH}{40 g NaOH}*\frac{1000mL H_{2}O}{10 moles NaOH}= mL H_{2}O$
• To prepare a 25 mL solution:
$25 mL sol. * \frac{800*10^{-3}moles PIPES}{1000 mL sol}*\frac{302.37 g PIPES}{1 mole PIPES}= '''6.0474 g PIPES'''$
$25 mL sol. * \frac{10*10^{-3} moles of EGTA}{1000 mL sol.}*\frac{380.35 g EGTA}{1 mole EGTA} = '''0.950875 g EGTA'''$
$25 mL sol. * \frac{1.25 moles NaOH}{1000 mL sol}*\frac{40 g NaOH}{1 mole NaOH}= '''1.25 g NaOH'''$
$25 mL sol. * \frac{10*10^{-3}moles MgCl_{2}}{1000 mL sol}*\frac{1000 mL sol. MgCl_{2}}{1 mole MgCl_{2}}= '''0.25 mL sol. MgCl_{2}'''$

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.