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DNA CONC.. Help?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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DNA CONC.. Help?

Postby abbydcruz » Mon Mar 08, 2010 3:44 am

Hi,
I have 15uL purified plasmid DNA, to which I added 285uL distilled water. The absorbance at 260nm was 0.163. Am i right in calculating the concentration to be 163ug/ml, by the formula 0.163x50x20=163? Or should i do it this way: 0.163x50=8.15, 8.15/300 gives 0.0271, 0.0271x1000 puts in back to mLs, giving a concentration of 27.1ug/ml; the second way apparently takes into account the concentration being ug/300mls instead of ug/ml. Please clarify..

Furthermore, if I need a 10ng sample of DNA, how do I dilute another 15ul sample so that between 1-10ul of the diluted sample equates to 10ng DNA?
Thanks in advance for your responses,
Abby
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Postby JackBean » Mon Mar 08, 2010 6:36 am

Hi,

there's no need to double your posts, you won't get answer faster.

If you want the concentration in the original 15 ul, than I vote for the first formula, because 0.163×50 tells you conc. in the 300 ul and ×20 is the conc. in original 15 ul.


Even the dilution you used gives you 8.15, so the question is, how much exactly you need it. But basically dilute it 16.3-times ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby abbydcruz » Mon Mar 08, 2010 8:56 am

Brilliant, thanks for clarifying the formula problem. Yeah, I ended up using a dilution factor of 32.6 so that I could say 2ul of the solution yields the 10ng of DNA needed. This required adding 474ul dH2O.

Also, another two samples of the DNA were boiled and treated with 0.1M NaOH, respectively. Their absorbance at 260 was also recorded, with the boiled DNA having about 0.03 higher absorbance then the NaOH. What could aspect of the denaturation could account for those discrepancies?

Thanks again for your help, sorry about the double posting thing, wasn't too sure where it belonged :)
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Postby JackBean » Mon Mar 08, 2010 11:22 am

look for DNA denaturation and melting temperature (of DNA) alias T_m (m is subscript)
http://www.biolib.cz/en/main/

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