I am getting very high 260/280 ratios after a phenol chloroform extraction of small sized DNA fragments. Extra care was taken not to leave any phenol or chloroform in the DNA solution before ethanol precipitation. Two chloroform extractions were done just to be sure not to have any phenol remaining.
Since the amounts of DNA I am dealing with are very low, I raised the volume of DNA with water before phenol chloroform extraction. This way I can leave a little aqueous layer above the organic layer in order to minimize DNA loss. But accordingly, the volume of Na acetate to be added for precipitation goes up. Could this be a problem?
Hi, Sanjay, you also should check A230 and measure both 2 ratios: 260/230 and 260/280. In the case of you have a high pick at 260 nm, I do not see any problem.
How high is very high for the 260/280 ratio? From what samples do you extract or purify the DNA? Are you sure that there is no RNA contamination present in your sample, as this will increase the 260/280 ratio.
Hi Christian, there is no RNA since I am purifying a ligation mix of two PCR fragments. High 260/280 I am seeing is something like 5-6.
Before ligations, the individual PCR fragments have no problems in the OD. This high OD problem is there with only ethanol precipitation also where no PCI was used, though ethanol should not absorb near 260 region...
I would suspect a Phenol contamination. See here: http://seqanswers.com/forums/showthread.php?t=21280
Hi Sanjay,
Ensure starting sample does not contain organic solvents (e.g., ethanol, DMSO), strong buffers, or alkaline reagents. These can interfere with phase separation.
Make sure to add chloroform that does not contain additives. After addition of chloroform, the homogenate must be vigorously shaken. If the phases are not well separated, shake the tube vigorously for at least 15 s, and repeat the incubation and centrifugation step.
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