[TI 84 Plus C]

I have to make a Resuspension buffer for a plasmid exaction, The buffer is based on Tris Base and EDTA, with RNAse A added.

The components are:

-50mM Tris-HCl (pH8)
-10mM EDTA (im using EDTA disodium dihydrate)
-100 microgram/mL RNAse A

I don't have stock solutions, all I have are the salts for each compound.


My plan was to first make a 50mL solution of 50mM Tris, then pH it to 8 with HCl.
Then make a 50mL 10mM EDTA solution, and add it to the Tris.

Then just add the RNAse A after.
But then I realized I would just diluting everything if I do it this way.

I really don't know how to start.
Any suggestions?

[Babcock_Hall]

Some general thoughts:  You may be able to make the stock solution of RNase A very concentrated  (10 mg/mL or even higher), in which case diluting it into the buffer will not change the composition of the buffer much.  Disodium EDTA is lower in pH than 8.  So if you could adjust the pH of the buffer in the presence of EDTA, I think that you will have better control over the final pH. In other words I would weigh Tris base (solid) and EDTA salt, dissolve in very roughly 75% of the water I think I might need (depending on circumstances), adjust the pH, then take this solution to the final volume of 50 mL.

[Borek]

I really don't know how to start.

Prepare stock solutions with higher concentrations and mix them so that the final concentration is the one you need, preferably going for concentrations so high that you will to dilute the mixture even further with water. This is not much different from what Babcock_Hall suggested, just measuring volumes is easier..

[TI 84 Plus C]

Thank you Babcock hall, I'm going to measure out 0.30grams of Tris, 0.19grams of EDTA disodium 2h2O, then dissolve them together in 37.5mL DI water.
I will then ph to 8 with HCl, then take it up to 50mL, and then finally add the rnase a.

Thank you for your reply Borek, can you send me links on learning how much of the concentrated stock solutions I need to add to reach my desired concentrations?
Sorry I am still an undergrad

[Borek]

Thank you for your reply Borek, can you send me links on learning how much of the concentrated stock solutions I need to add to reach my desired concentrations?
Sorry I am still an undergrad

All you need is high school level dilution calculation, see for example http://www.chembuddy.com/?left=concentration&right=dilution-mixing

[Babcock_Hall]

Assuming that your RNase A is a lyophilized powder, you can add that to portions of buffer just before use.  It will have a negligible effect on the volume.

[TI 84 Plus C]

I am actually worried that the RNAse A powder may contain DNAse

[Babcock_Hall]

At least some DNases require a divalent metal ion.  I would predict that they are less active in the presence of EDTA.

[TI 84 Plus C]

Thanks for all the help so far!
Any suggestions on what concentration HCl I should pH the buffer with?

Today I made a 100mL 3.0M Potassium Acetate solution that I had to go to pH 5.5
The solution pH was 8.5 when first made, but by the time I finally got to pH 5.5 by adding HCl, the final vol was 200mL.

So it took me 100mL of acid to pH my solution, which means it is no longer near 3.0M
I used 1"N" HCl, but added a couple micro liters of concentrated stock HCl every now and then.

[Borek]

Today I made a 100mL 3.0M Potassium Acetate solution that I had to go to pH 5.5

Potassium acetate is just a solution of a base. If you need a pH 5.5 buffer you should prepare it from acetate and acetic acid (or at least use highly concentrated HCl to get close to the final pH first). It is relatively easy to calculate amounts of acid and base needed to make preparation easier, compare http://www.chembuddy.com/?left=buffers&right=toc Note it doesn't mean you should use highly concentrated HCl every time, but as a rule of thumb if you use acid (1 M in your case) that is much less concentrated than the original solution (3 M as you wrote) you will be diluting first, changing pH second.

Acetate buffer is not part of your original mixture, I guess these things are unrelated?

[Babcock_Hall]

I agree that doing a scratch calculation first is extremely helpful.  Also I would pay attention to the ionic composition of your buffer; some biochemical phenomena will respond to differences in ionic strength or to the counterion.  In this particular case if you use both acetate and acetic acid, the concentration of the buffer is described in terms of the sum of the concentrations of acetate and acetic acid.  One relatively easy way to make an acetate buffer is to make stock 1 M solutions of acetate and acetic acid and then mix them to obtain the desired pH.

When making a Tris or glycine buffer, I sometimes start with concentrated HCl or 6 M HCl, then step downwards to 3 M or to 1 M as I approach the desired pH.  This lessens the chance that I will overshoot.  The exact concentration is dependent on the concentration of base.