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ratios insert/vector

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#1 lucilius

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Posted 20 October 2014 - 12:53 AM

Dear all,

I am a bit confused about the ratios conceding vector/insert...

If I check this website: http://www.addgene.o...s/dna-ligation/ , I can see they use 25ng vector and 75ng insert.. This is a ratio of 1/3 (vector/insert).

And yes: this is a normal ratio, however, it seems to make no sense if you use calculators like this one: http://www.insilico..../Lig_Input.html

The amount (in ng) of oligo (insert) that has to be used for cloning is always a lot lower than the amount of vector (in ng).

Lets say: we have 5000 bps of vector, 25 ng    and we have an insert of 500 bps... This calculator then tells you to use 7,5 ng insert!!

This is not the 75 ng ...

I do not get this.

Is the addgene website a bit mistaken and taking ng ratios for molar ratios?

#2 phage434

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Posted 20 October 2014 - 05:30 AM

1) The ratios matter far less than most people imagine. I clone routinely without ever thinking about them.

2) The ratios are molar ratios. 7.5 ng of a 500 bp insert will have 3x as many molecules as 25 ng of a 5000 bp vector.

#3 lucilius

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Posted 20 October 2014 - 09:45 AM

1) The ratios matter far less than most people imagine. I clone routinely without ever thinking about them.

2) The ratios are molar ratios. 7.5 ng of a 500 bp insert will have 3x as many molecules as 25 ng of a 5000 bp vector.

Regarding point 2: than I do not understand the addgene website.

I understand your answer and the programs (found online) to calculate the ratios, but the addgene website just states 75 ng insert and 25 ng vector. Makes no sense since the vector is larger than the insert so less molecules than the smaller insert.

#4 phage434

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Posted 20 October 2014 - 06:59 PM

I see what you mean. I hadn't looked at that page. Short answer: they got it wrong. I'll have them fix it.