Buffer with multiple ingredients

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Buffer with multiple ingredients

Postby learner1 » Jun 02 2017 10:37 am

Hi,
I got a modified CTAB protocol and I need to prepare the following Nuclei extraction buffer:
10 mM TRIS-HC1 pH 9.5,
10 mM EDTA pH 8.0,
100 mM KC1,
500 mM sucrose,
4 mM spermidine,
1 mM spermine,
0.1% beta-mercaptoethanol).

And the protocol says, add 10 ml of ice-cold Nuclei extraction buffer.

I am unable to figure out how to prepare the buffer solution. I need to prepare 500ml.
Thanks in advance and look forward to your feedback please.
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Re: Buffer with multiple ingredients

Postby relaxin » Jun 03 2017 10:09 am

First find the molecular weight for all the chemicals (should be on the label). Then calculate the amount of each chemical you need for 500 ml of solution.

For example, 10 mM Tris HCl is 10 mmoles of Tris HCl in 1 liter of solution. Since you are making 500 ml of solution, you need 5 mmoles or 5 x 121.14 g/mole (mw of Tris HCl) = 605.7 mg of Tris HCl. I think you can figure out the amount for other chemicals.

Dissolve all chemicals in about 400 ml of water, adjust the pH and make up the volume of 500 ml with more water.
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Re: Buffer with multiple ingredients

Postby learner1 » Jun 04 2017 6:05 pm

Thanks a lot relaxin for your response.
I could calculate the mM concentrations but how to adjust two pHs for two solutions? For example 10 mM TRIS-HC1 pH 9.5 and 10 mM EDTA pH 8.0.
Best
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Re: Buffer with multiple ingredients

Postby relaxin » Jun 04 2017 7:33 pm

I thought this is one buffer with multiple ingredients. What is the final pH of the solution?

EDTA is difficult to dissolve, unless you bring up the solution to pH 8.0 with NaOH. You can make it more concentrated (say 100 mM), so that you can add 50 ml of the 10X EDTA to the final buffer prior to the adjustment of final volume to 500 ml.
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Re: Buffer with multiple ingredients

Postby learner1 » Jun 05 2017 9:42 am

It says,
10 mM TRIS-HC1 pH 9.5,
10 mM EDTA pH 8.0

This is somewhat confusing.
Thanks so much.
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Re: Buffer with multiple ingredients

Postby learner1 » Jun 05 2017 10:12 pm

The protocol does not mention a final pH of the solution. The extraction buffer is as follows:
10 mM Tris-HC1 pH 9.5
10 mM EDTA pH 8.0
100 mM KC1,
500 mM sucrose,
4 mM spermidine,
1 mM spermine,
0.1% beta-mercaptoethanol

Thanks again relaxin for your input.
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Re: Buffer with multiple ingredients

Postby relaxin » Jun 06 2017 10:12 am

I checked a number of nuclear extraction buffer recipes available online. Most of them use pH 7.8 to 8.0. I guess you can adjust the final pH of your buffer to pH 8.0. pH 9.5 seems to be too high.
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Re: Buffer with multiple ingredients

Postby r.rosati » Jun 07 2017 11:10 am

Hi!
Just dropping by to write that it's "KCl" and "HCl", not "KC1" and "HC1".
"Cl" is the chlorine element.

(Same as "H20" is "H2O")
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