Calculating the number of plasmid molecules

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robotnick
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Calculating the number of plasmid molecules

Post by robotnick » Fri Apr 24, 2009 9:03 pm

Hello,

Any help on this question would be appreciated.

If the average plasmid size in the ligation mixture = 3kbp, how many plasmid molecules were in tube A? Assuming that the average MW of a nucleotide = 320. The amount of DNA we were given in tube A was 1 ul (concentration 0.1 mg/ml).



- I believe the mass of an average plasmid would be 192 x 10^4 [considering it is double-stranded - 6kbp], can't get any further...

blcr11
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Re: Calculating the number of plasmid molecules

Post by blcr11 » Fri Apr 24, 2009 11:52 pm

You have one of the numbers you need, but you kinda got the right answer for the wrong reason, and you haven't determined the mass, you've determined the molecular weight in gm/mole of the 3000 bp plasmid.

You've been gven the molecular weight of an average nucleotide (320 gm/mole), but the plasmid is 3000 base pairs. You didn't suddenly acquire 3000 more bases; the plasmid is still 3000 bp long, but the average MW of a base pair is 2x the average MW of a base. See what I mean?

So, the calculated MW of the plasmid is (2x320 gm/mole bp)x3000 bp = 1.92 E4 gm/mole.


The mass of plasmid in 1 ul of a 0.1 mg/ml solution of plasmid is 100 E-9 gm [ 0.1 mg/ml = 0.1 ug/ul = 100 ng/ul ]

The # moles plasmid then is 100 E-9 gm/1.92 E4 gm/mole = 5.2 E-12 mole or 5.2 pmole.

jgreggain
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Re: Calculating the number of plasmid molecules

Post by jgreggain » Thu Feb 25, 2010 3:22 pm

blcr11 is mostly correct. I'm not sure how, but something go messed up in the E values.
The calculated MW of the plasmid is actually 1.92 E6 g/mol (vs 1.92 E4).

This then equates to having 5.2 E-14 mol, or 52 fmol of plasmid (vs 5.2 pmol).
So, the numbers are correct, but they ended up being off by a factor of 100 somehow...

Hope that helps!

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