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# preparing buffer

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### #1 claritylight

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Posted 23 March 2005 - 12:34 PM

This is my stock:
1M Tris-HCl
1M CaCl2
1M MgCl2

This is what I want in my 10X reaction buffer:
50mM Tris-HCl pH 2.5
10mM CaCl2
10mM MgCl2

I want the total volume of my buffer to be between 3-5mL so if I mess up when using the buffer, I'd still have some left over.

I found the dilution factors for each, so for example, for the Tris-HCl:
1000mM/50mM = 20.
Then I took the total volume/dilution factor to find out how much Tris-HCl I would need: 5mL/20mL = 1/4 mL Tris-HCl

For CaCl2: 1000/10 = 100 fold dilution factor
5mL / 100mL = 1/20mL CaCl2 (same amount for MgCl2)

Then, I added them up and subtracted by 5mL to find how much water I needed to add:
1/4 + 1/20 + 1/20 = 7/20 reaction solutions
5- 7/20 = 93/20 mL H2O

Would doing it this way get me the 10X buffer I need?

Edited by claritylight, 23 March 2005 - 12:34 PM.

### #2 fred_33

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Posted 23 March 2005 - 01:56 PM

yep you're right

### #3 tfitzwater

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Posted 23 March 2005 - 03:02 PM

There is a short cut to calculating the amounts required. If you are starting with a 1 M stock and want 50 mM final, you need 50 uL per mL of final volume. For 10 mM, you need 10 uL per mL of final volume. So for 5 mL, you need 5 x 50 uL of 1 M Tris and 5 x 10 uL of 1 M CaC2 (and MgCl2) respectively.

### #4 claritylight

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Posted 23 March 2005 - 05:39 PM

There is a short cut to calculating the amounts required.  If you are starting with a 1 M stock and want 50 mM final, you need 50 uL per mL of final volume.  For 10 mM, you need 10 uL per mL of final volume.  So for 5 mL, you need 5 x 50 uL of 1 M Tris and 5 x 10 uL of 1 M CaC2 (and MgCl2) respectively.

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This is good. I usually convert the mL to uL (since we usually use the uL pipettes), but this way really speeds it up.

Thanks all.