I have to extract DNA from lung ffpe samples with pure link invitrogen kit and high pure PCR template preparation kit Roche. I use 20 ul 20mg/ul RNase A for each sample (about 50 mg tissues).The ratios A260/280 = 1.8 - 2.2, A260/230 >1.8, but the image of gDNA on 1% Agarose gel has smear from the top of gel. I dont know the reason, whether there are still RNAs or some inhibitors through extraction procedure??
hello
it looks like degraded genomic DNA , is your kit not expired yet? , if you want to perform a simple end point PCR; this will make it, but if you want sequencing or qPCR standards this wont help
this looks like good quality and randomly degraded dna as expected from old samples. the rna content is low as shown by the 260/230 ratio. If the next stage is pcr then there is nothing wrong with high molecular weight degraded dna which will amplify well. Actually being a bit degraded it will melt better than very high MW dna so will amplify better.
Hi,
All of the possibilities and suggestions have been already discussed. I just want to mention a little more, seems like the quality of Agarose Gel was not that good if you look into the Ladder you will find. I think you could have mentioned the staining and de-staining duration and the EtBr conc. were being used. In case of gDNA, you were supposed to get a thick band near the well, though i am not sure about the concentration of gDNA. So suggestion might be like that you should run the sample in agarose gel considering all these.
I repeat as like above that the purity of gDNA is good and if RNase treatment is good there should be no chance of finding RNA.
Best,
Imtiaz
I would say if everything else is checked as previously explained above, i would, as a last resort check your DNA gel, It looks like all of your wells are smeared and even your DNA ladder is smeared and your ladder shouldn't be.
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