I am experiencing a contamination problem in my DNA extracts. I did DNA extraction from water samples using phenol/chloroform. However, I do get a shoulder/high peak at 230 nm whereas there is a slight peak at 260 nm when measured with Nanodrop. My 230:260:280 ratio is more like this: 1.6:0.8:0.5.
I adapted my fieldwork sampling protocol from Foote et al. (2012). Based on the literature search, and similar problems people have experienced, I do think that the high peak at 230 nm is due to some salt contamination along with residual ethanol. I am in the midst of troubleshooting, but I am not sure how to fix this problem. There are people suggesting to use ether (to remove organic solvents) or to dialyse the probe if salt is the contaminant. Also, during PCR some additional reagents can be used, such as BSA and/or glycerol to prevent any potential inhibitors getting amplified.
Any suggestions on how to carry out? I would like to have purified DNA before troubleshooting with PCR step, ideally.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0041781
Starting with the quickest and easiest things I can think of:
If it's residual ethanol, you can often just warm the samples in the water bath at 50C for 30 minutes or so, and the EtOH will evaporate.
If it's residual organics, a second extraction with straight chloroform will help remove them.
Finally, a second precipitation can't hurt (add 1/40th volume of 5M NaCl and 2x volume EtOH, let precip for an hour, spin max speed for 10 minutes, then wash with cold 75% EtOH) -- It can dilute out some other salts that come along for the ride.
Respected madam
For DNA Extraction phenol-chloroform is good method no doubt about it , but way of doing is matters, like when we are collecting supernatant (because it was liquid - liquid separation) , so as per my knowledge Kit method is useful and contamination free ( Like I use QIAGEN KIT Protocol) for good 260/280 ratio.
260/280 ratio likely around 1.2 we have to get.
All the best
Anil
I suggest you have problems with pcr inhibitors such as fumic and humic acids. Theses component are found from environmental samples (especialy in stagnant water). These are axtracted with dna in standard extraction protocols. Try some specific kits or protocols to remove these chemicals.
Thanks for the very helpful suggestions. I did carry out a purification step using Qiagen kit on my samples. After this step, I was able to get rid of the peak at 230 nm. It just seems to be a time-consuming way of getting the clean product out, but I assume that as @Mathieu Dondelinger mentioned, there may be some acids that are co-extracted with DNA for this particular fieldwork site. As long as I get the clean products, I think it should be the way to go for now.
I have experienced similar issues with high A260:A230 ratios for DNA (samples ranged between 2 and 10). If the A280:A260 ratio is 1.8 then this indicates a low absorbance at 230nm.
If you experience a low A260:A230 ratio then pellet the DNA and wash it with ethanol, and allow time for the alcohol to evaporate off before adding elution buffer. Read the same again and the A260:A230 ratio will increase. Deliberately add some ethanol to a dummy sample and a decomposed pcr mix to another dummy sample and read them to observe the effects to gain confidence in the high A260:A230 readings.
Regards,
Robert.
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