RNA contamination in spec analysis

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astha
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RNA contamination in spec analysis

Post by astha » Thu Nov 17, 2005 4:52 am

hi everyone

i am a new member to this forum and want some help relating to plant DNA extraction. I add RNase in the last step of my protocol, thus prediting the interference of protein in the dissolved DNA. But still the spectrophotometric readings are 1.88 - 2.01 at absorbance at 260/280. Why this is happening I am not able to understand, I even increased the concentration of RNase, but it did not change

Astha

sdekivit
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Post by sdekivit » Sat Nov 19, 2005 4:40 pm

you have no contamination of RNA --> a pure DNA-sample has a value of 1,8-2 at a A260/A280-raTIO ;)

astha
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Post by astha » Thu Dec 08, 2005 8:42 am

thanks :)
i was wondering if i am missing right in the beginning.
now i can think of PCR amplification of the plant using RAPD.
do you hav any suggessions.

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Your DNA extraction was good, Astha

Post by Jumpshooter » Wed Dec 14, 2005 7:32 pm

you got a26/280 ratio of greater than 1.5 indicating less protein contamination; however, I'd suggest you also run a picogreen assay to determine exactly how much dsDNA template you have in that sample. The a260-280 is telling you the Total level of nucleotides (rna, ssdna, oligonucleotides, free nucleotides, and ds DNA). Remember, it's only the dsDNA fraction that is an amenable template for PCR!

Bon chance!

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