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What is meant when asked write molar ratios in 3 parts? For example I am currently assembling a vector that requires annealing several oligos to two pcr fragments prior to ligating them into my vector; which will be used for future transformations. I was told to "assemble the annealed oligos with the 2 pcr fragments. Use a 100:1:1 molar ratio of annealed oligos:each pcr fragment." I'm thinking this means- concentrated oligos:diluted pcr frag #1:diluted pcr fragment #2. I am confused about how to determine the final concentrations of my pcr fragments in relation to the concentrated/undiluted annealed oligos. Why? Because I only know their concentrations as ng/uL not as 5x, 2x or 1x. Should I calculate it as 1(part) divided by 102 (whole) for each fragment? Then multiply that number by the actual concentration of my pcr fragments.

I am sure the answer to this is probably simple but I'm sorry I don't understand it and am hoping someone helps me with understanding calculations of this sort. Thanks.

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The whole point of this problem is that for each 100 molecules of annealed oligos, you need to have one molecule of each fragment.

You need to take into account the following:

The MW of each fragment is approximately proportional to the length of the fragment. Thus if you divide the concentration by the length of each fragment, you can get a value that simulates the molarity, meaning that you can use these values to fix to the ratio that you are asked between the different fragments.

For example, if the fragment1 is 100 bp and you have 10ng/µl, fragment2 is 200 bp with the same concentration, then you need to add 1 volume of fragment1 and 2 volumes of fragment2 to get the 1:1 molar ration.

In a similar way, you can calculate the amount of oligos added to have 100x more (of the ratio of concentration to bp length) than the respective fragments.

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