Gradient preparation - (Oct/10/2011 )

Hi,

I am currently trying to follow a protocol in producing a sucrose gradient in order to ultracentrifuge serum into various lipoproteins. However the step my step instructions seem alittle abscure for me. If anyone can shed any light as to how to follow the various steps it would be much appreciated.

The two steps I am having difficulty to interpret are:


"5.0mL of serum (adjusted to 4.0M in NaCl by adding solid NaCl directly to serum"

"9.0mL of a 0.67M NaCl solution containing 5% EDTA, pH 7.0"

I understand from the above that i need 5.0mL of serum, but i need to adjust to 4.0M in NaCl?

As to the second passage i can calculate as to how to get 9.0mL of 0.67 NaCl but how much 5% EDTA do i add, and then how to i get it to pH 7.0??

Thank you all.

You know how to make solutions I am presuming - so in your first part - make the 4 M NaCl using the serum as diluent instead of water. You may want to find out the NaCl concentration in the serum first to make sure that you make the final concentration to be 4 M

For the second part you will probably find it easiest to make a 0.5 M solution of EDTA, pH this to 7.0, and then add some of this to the NaCl to make 5% EDTA. It is quite common in biology to make a solution to a particular pH and then dilute it into another solution, so you will see things written like: XX mM EDTA, YY mM Tris-Cl (pH 7.5), ZZ M NaCl.

Thankyou.

I have managed to work out how to make my sucrose gradient from the following protocol that I am following:

http://www.thermo.com/eThermo/CMA/PDFs/Various/File_6808.pdf

The next step is to make the 5% EDTA. Im sure its simple enough once I have read through my research.

Thankyou for your help.