volume of my samples (~30uL) into the gel in order to see the band although the concentration of DNA
Have you removed RNA from your sample? Are your DNA concentration estimates based on spectrophotometric measurements or another assay? If you isolate genomic DNA, you should not see a single band, and due to its high molecular weight, it may be difficult to get to migrate through a typical agarose gel. It is possible that your DNA simply doesn't enter the gel very efficiently.
what is 260/280 ratio of your sample....if its some where near 1.8 then ok, else u purify your DNA . what percentage of gel you r using...use .8% of gel.
no need to load too much volume of sample due to the concentration is high. You can load in your sample in range of 3-5 ul. that's enough to see your isolated genomic dna and supposed not to have a far migrated band down the agarose gel because of high MW and size. You can compare the size by using lambda HindIII for determination of genomic dna size.
you are loading 30 ul that means your are preparing a thick gel, thickness of the gel may reduce the intensity (transmittance)of uv light under uv transilluminator. other factor that may be consider are agarose conc., purity of DNA. You can run lambda DNA or any other control DNA to ensure that.
I think my DNA sample is not pure enough due to the humic acids. By the way, the concentration that I use is 0.7%. May I know how the conc of agarose gel affects the visible of band?
The concentration you stated of 1000 ug/ml DNA = 1000 ng/ul. You state needing to load more than 30 ul to see a band. 30 ul of 1000 ng/ul = 30,000 ng or 30 ug. On an agarose gel you should be able to see 50 ng with EtBr staining easily. By extrapolation your A 260 readings are incorrect (over estimated). If you cannot even see RNA, then the A260 contribution is due to something else. Reprecipitate your samples and resuspend in as small a volume as is useful for downstream needs.
One last thought, how long after final resuspension do you wait before analyzing your samples? If you are resuspending in water, it is very possible that you are losing your sample by degradation prior to loading. Since you are running a low percentage gel which is used to resolve high molecular weight DNA you will not see a HMW DNA band. Instead, you will see the smear of degraded DNA.
with respect to the concentration of agarose, it is chosen based on size fragments you which to resolve. There are some excellent chapters and books on DNA electrophoresis available.
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