3 replies to this topic

[bioduud]

Hi can someone please help me with a math problem?

I have a miniprep ( purified plasmid) concentration 225 ng/µl of a plasmid sized 2505 bp.  I want to start a 10µl reaction that contains 0,001 µM DNA. Is my math correct:
so i found this :

 

DNA/RNA molarity Calculator:
  M = w / MW
Where:
M: DNA molarity, in mol
w: DNA weight, in g
MW: DNA molecular weight, in g/mol

If only DNA length is given, the molecular weight is calculated as:
MW = DNA Length (bp) × DNA/RNA base weight
The approx. double strand DNA base weight is 650 Dalton, for single strand is 330, for RNA is 340 Dalton

therefore for 1 M reaction  2505*650=1628250 g/l??.
but I'm confused of how to continue my calculations?

Edited by bioduud, 17 July 2015 - 12:17 PM.

[phage434]

Well, to start, molarity is not measured in moles, but in moles per liter. So M is moles, not molarity. It is important to keep these things straight.

You can see this from your equation, if you substitute units:

 

M(moles) = w [grams] / MW [grams/mole]

 

Note that the grams cancel, and the mole moves upstairs. Liters appear nowhere.

 

Now, to get back to your question. You may wish to calculate the MOLARITY of your solution of DNA. A similar formula can be used:

 

molarity [mole/liter] = concentration [g/liter] / MW [g/mole]

Check the units: grams cancel, moles move upstairs, liters remain.

 

Substituting:

molarity[mole/liter] = 225 ng/ul  /  1628250 [grams/mole] = 225e-9 g/ul  / 1.63e6 g/mol = 1.38e-13 mol/ul = .138e-6 mol/liter = .138 uM [mol/liter] = 138 nM [mol/liter]

 

Now, your reaction needs .001 uM (this is independent of volume). To achieve this, you need to dilute your DNA from 138 nM to 1 nM, which is a dilution of 138 times. You can't readily achieve this in a single dilution step, so you likely want to first make a 10 nM solution by diluting your DNA 13.8 times (for example, 1 ul of DNA into 12.8 ul of water). Then, your final reaction setup would add 1 ul of your diluted DNA  to 9 ul of whatever other reaction components were necessary to create a final 10 ul volume.

[bioduud]

Thank you for your very informative and fast answer, you saved my day!

Edited by bioduud, 17 July 2015 - 02:11 PM.

[Leslieter]

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