Agarose gel electrophoresis, I have ran (2ul sample+1ul 6X gel loading dye+ 3 ul sterile water) plasmid (6kb) sample in 1 % agarose (Low EEO grade) gel with Zip Ruler Express DNA Kb ladder ( ladder details 100,300,500,850,1200,2000,3000, 5000 and 10000), after the run I checked the gel doc, the ladders are fine but there is no band of plasmid. When I checked the absorbence it is 400ng/ul (A260/280) 1.3. please help in solving the problem.
Absorbance readings are not very reliable for determining DNA concentration unless the sample is relatively pure. Anything absorbing at 260 nm (free nucleotides, primers, RNA, etc.) will bias the measurement. In addition, at that concentration degraded DNA may produce a smear so faint you might not notice it (depending upon the size distribution of the resulting fragments).
If you will add picture then it will be more helpful to see whether it is showing smear or not. or else you may try with increase the sample volume (i guess 3ul).
I agree with Alejandro that a lot of things might absorb at 260 nm, so there is no way to spectrophotometrically assert that the absorbance came from DNA. You might quantify dsDNA by fluorescence spectroscopy if available. Also, strange choice of 100 bp ladder for targeting 3 kb plasmid. You might want to use some broader middle-range ladder, but this is no answer to your question. If plasmid is commercial, I'm sure it would work if you increase the amount of DNA you load on the gel as Harmesh proposed and maybe to increase the concentration of EtBr, or any other fluorescent agent you use.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques