Hey all,
I get puzzle in a simple calculation.
I have a protocol for qPCR, according to instruction i need 5ul of DNA having the concentration of 1ng/ul, for starting a standard curve. My DNA stock solution have a concentration of 176ng/ul. How should i adjust this concentration, so that i get 1ng/ul in 5ul ???
Thanks,
Regards.
Hi there,
So you need 5ng of plasmid in your final mix, right? It's a matter of dilution: you have a 176ng/µL solution you want to turn it into a 1ng/µL so you have to dilute 176 times. Depending on how much you want to engage in the process you may put 1µL of the sample in 175µL of buffer or more generally xµL of sample in 175x µL of buffer.
Hi Waqas, just out of the box in another way you can solve it. You want 1 ng / ul in 5 ul, which means 5 ng / 5 ul (as your each ul contains 1 ng). So to solve it;
If you have 176 ng in 1 ul
So, 1 ng in 1/176 = 0.00568 -1 = 0.9943 (diluent you need), but to take 0.00568 ul sample from your stock is impossible by any pipette and also if you add 0.9943 diluent into this so it will never make 5 ul. So for such values multiply by any factor, which can give you decent amount in hand, which can also be picked through pipettes and in the end you have good volume for successive experiments as well.
So now for example; 0.00568 x 500 (which is purely my assumed factor) = 2.84 ul (so that I can reach to a certain volume which can be easily picked by pipettes)
Same factor you need for the diluent too (water or buffer) hence 0.9943 x 500 = 497.15 ul
So just take 2.84 ul from your main stock of 176 ng / ul and add 497.15 ul diluent and you have then 1 ng in 500 uL total volume and just take 5 ul of it for your work and safe the rest. So every ul now has 1 ng in it.
Now to check this, simply divide 497.15 / 2.84 = 175, which is your dilution factor, means your calculations are right. In above example you can play with any other factor and in the last you will get the number 175. Let's say multiply 0.55568 and 0.9943 with 700 and then follow the above steps, finally you will get 175.
Regards...
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