User:Brigette D. Black/Notebook/Brigettes Notebook/2009/07/10/POP-ing Buffer
x2 POP-ing buffer
- 50mM phosphate (dibasic/monobasic ratio to be pH 7.5)
- 2.5 mL KCl at 4M
- 20 mL EDTA at 100mM (pH 8)
- 0.8 mL Tween at 5%
- 156.70 mL DI H2O
As we did not have any dibasic sodium phosphate, I use potassium phosphate and KCl where once sodium was needed.
- Monobasic Potassium Phosphate( Fisher Chemical P285-500)
- Dibasic Potassium Phosphate( Fisher Chemical P288-500)
- EDTA ( Aldrich 431788-100G)
- Sodium Hydroxide (Fisher S318-500)
- KCl (Fisher P217-500)
- Tween (origins unknown)
pH-ing Potassium Phosphate
I made a 500 mM stock of each by weighing out 1.8787 g and 1.443 g of dibasic and monobasic phosphate respectively. To these I added 12.6 mL and 21.2 mL DI H2O.
In order to get the monobasic and dibasic ratio to be pH 7.5, I found a table and found that we needed a ratio of 4.882 diabasic to monobasic. So, I added 3.4 mL monobasic stock to 16.6 mL dibasic stock into a glass jar. Note here, if we were using sodium phosphate, the ratio should be 4.26 dibasic to monobasic.
Making 100mM EDTA
EDTA will not so into water at 100mM, so we have to force it to do so by adding little bits of sodium hydroxide and stirring. The pH of the solution where 100 mM EDTA is reached should be about 8.
We weighed out 2.924 g EDTA and added this to about 80 mL DI H2O. Lihn then set up the pH meter so that we could tell when the solution got to pH 8. Lihn and Koch began by adding a stock solution of sodium hydroxide, but when this was going very slowly, they began just dropping in pellets. The end result was a 99 mM solution at a pH of 7.8.
Mixing it All
The final recipe (as it was made) is as follows:
- 3.4 mL Monobasic Potassium Phosphate (at 50mM)
- 16.6 mL Dibasic Potassium Phosphate (at 50mM) (final Phosphate: 50 mM)
- 2.5 mL KCl (at 4M) (final: 50mM)
- 20.2 mL EDTA at (99 mM) (Final 10mM)
- .8 ml Tween at 5% (final 0.02%)
- Total Volume: 200 mL
All of these were mixed in a 250mL glass Wheaton jar and is now stored on the shelf above my workbench.
- x1 Pop-ing Buffer
This is made by adding an equal volume of DI H2O to 2x Poping buffer.
- BGB+ x1 Pop-ing Buffer
This is made by making a 10 mg/ml BGB solution in x1 Poping buffer. I weighed out 50mg BGB and added 5 mL of the x2 Pop-ing buffer. I then filtered this through a 02 micron filter to get out an undissolved bits. (Steve Koch 22:37, 14 July 2009 (EDT): And another purpose of the 0.2 micron filter (more important) is that no bacteria can make it through that size filter. Thus it's a quick way of sterilizing the solution. It's also a good way of sterilizing when you cannot autoclave (proteins cannot be autoclaved w/o denaturation).) DI H2O. This solution is needed when using microspheres and tethering DNA.