I extract DNA from liquid manure (Powersoil DNA Isolation Kit from MOBIO) but I constated that if I treat my sample by centrifugation twice as long at 14000 rpm I have a better yield (three time higher), which is expected because this way bacteria are more concentrated and I am able to get more DNA. I washed the sample with deionised water between the two steps of centrifugation. But if I compare the ratio of absorbance A260/A230 (Nanodrop) between the sample treated by centrifugation and those with no treatment I can see that the second have a two factor less than the first. My question is why this difference? Does the treatment I've done remove types of contaminants such as peptides, phenols or guanidinium (the extraction kit also contains guanidinium) or is it just due to the nanodrop assay method I used (I triplicated the samples).
High absorbance at 230 is usually guanidium, which as a chaotropic salt is....not good for any downstream reactions.
You can usually remove excess salt by simply ethanol or isopropanol precipitating your samples again. If your 260/230 ratios are lower than 1.75, I would certainly reprecipitate, and if you've got a lot of DNA (so you can afford to lose some) I would consider re-cleaning any sample with a 260/230 ratio lower than 2.0.
A simple ethanol precipitation would be:
Add H20 to final vol of 500ul in a 2ml tube
Add 50ul of 3M sodium acetate, pH 5.5
Add 1.5ml of ice cold ethanol, incubate at -20 overnight of -80 for an hour
Collect by centrifugation 13krpm, 10 mins, 4 degrees
Wash twice with ice cold 70% ethanol (add 300-500ul 70% ethanol, spin, remove, add a second 300-500ul 70% ethanol, spin, remove)
Air dry, redissolve in H20.
A simple isopropanol precipitation would be:
Add H20 to final vol of 500ul in a 2ml tube
Add 50ul of 3M sodium acetate, pH 5.5
Add 500ul of ROOM TEMPERATURE isopropanol, incubate at room temp for 10-20mins
Collect by centrifugation 13krpm, 10 mins, 4 degrees
70% ethanol wash and so on (as above)
thanks you Mister John,
do you think that guanidium may inhibited completely a PCR downstream application?
Sorry i am not getting your question...
but if you are asking about the extraction procedure will give the same results ... theoretically it should give the same results if we are using the same chemicals and sample.... but practically it requires lot of experience as it not possible to give the same conditions in two tubes....
Could you please indicate the kit used?
I've been extracting DNA from extreme environmental samples using the FastDNA SPIN kit for soil and the Nanodrop is always useless with this kit or the equivalents from MOBIO. For a extreme case, I'm getting A230 of up to 15, yes 15, so actually you only see one peak in the spectra, with the A260 being covered by the "tail" of the A230 peak. With about 30 samples all the A260, A230, A260/A230, etc are always nearly the same but the PCR work very different depending on the sample: some working with our standard lab conditions (just 0.2 U Taq and 1 uL DNA) while others need up to 1 U Taq and 2 uL DNA for a very weak amplification. And no, no problem with inhibitors: tested by dilutions and spiking the samples.
Also, if you are extracting from liquid, you need higher speed and longer time to have a more representative population in your pellet. Check the publication below.
Article The recovery of nucleic acid from biomining and acid mine dr...
I read the publication that you refer. I did the same adaptation of my extraction protocol
(Powersoil DNA Isolation Kit). About the application of PCR I had been able to resolve the problem by rising the number of cycle (from 32 to 50) to have a PCR product. I will follow the investigation by rising the Tap Pol concentration as well as you mentioned. Thanks you again for your HELPS
(1)dont look just at 260/230 ratio, analyze the entire spectrum - Nanodrop or other instrument (2) dont worry much about this ratio, it does not mean much. I've seen some DNA samples with great 260/230- and PCR was bad, and vice verse- crappy 260/230 and great amplification. all that matters is good PCR downstream
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