Autoclave PBS-A with Na2HPO4 = cloudy? - (Nov/18/2009 )

I just started to prepare PBS-A. My PBS-A recipe came from Freshney's cell culture text book. However, I discovered that after i dissolved the mixture in water and autoclave, the PBS-A became cloudy with precipitation.

I'm sure the recipe is ok, as most of other protocol available online also suggested the same. The only modification I made is I used 1.14g Na2HPO4 anhydrous instead of the one suggested (not in anhydrous form).

Could the anhydrous form cause the problem? Is there anyway to solve it?

I compared my recipe with other labmates, and found out that they used NaH2PO4 instead of Na2HPO4. I am thinking if I can use their protocol, then adjust Na+ ion concentration using NaOH? Assuming the pH will be quite alkaline, then I will just adjust the pH back to 7.4 with HCl. Is that ok?

dcch on Nov 18 2009, 06:26 PM said:

There is a big difference between NaH2PO4 and Na2HPO4. One is acid and the other basic, you mix them to arrive to the appropiate pH.

I remember one of them fall out of solution when frozen, thus changing the pH, the same probably occurs autoclaving. If you pretend to freeze or heat strong switch to 50mM Tris-Hcl.

To prepare PBS
1)Prepare NaH2PO4 solution 0,5M
2)Prepare Na2HPO4 solution 0,5M
3)Mix (1)&(2) to obtain a stock of 0,5M PB with pH 7,2 (6,8-7,6).
4)Make a 5M stock of NaCl (a litle heat will make it dissolve fast)

To prepare a 20X PBS stock:

5)Mix 400 ml of (3) with 600ml of (4)

To prepare 1x PBS:

Mix 50ml of 5 with 950 ml of distilled water. Ready to use.

This is the standard PBS: 10mM phosphate with 150mM salt.

To prepare 1X PBS you can use distilled water. For the stocks use miliQ water and store them in fridge to avoid fungal grow.

We used to have the same problem.
What we did is instead of autoclaving the PBS, we run it throw a vacuum 0.2 micron filter to sterilize it.

Carl took a good look at the large stainless steel machine that stood in front of him. The pipes that supply the steam come down from the ceiling. He could see the insulation that covers the pipes was coming off in spots. The stainless steel panels were dented and cover with dust and dirt. Puddles of water cover the floor around the machine from the dozens of leaks in its pipes.

 instyler