the concentration of DNA sample before extraction is 4.16 u gm/ml, after extraction of this DNA from agarose gel the concentration was found as 4.15 u gm/ml hence,the recovery is 99.7%. But, after running this extracted DNA I got smear on the gel rather than clear blue bands. So, can u give me some suggestions to avoid these flaws.
Your DNA seems to be degraded by endonucleases, which won't affect its bulk concentration. Try using disposable, sterile plasticware or else autoclaved glassware and autoclaved buffers
probably , there is some RNA or protein in your extraction , or your can wash your pellet exactly to remove pollution.
next provide supernatant with better solution.
the pollution causes your DNA to be degraded or when extracting you could avoid pipetting fast.
Definitely sounds like an endonuclease degradation. Try using DNase free, sterile (autoclaved) tips and buffers.
I agree with the above and mention also that vortexing rather than inversion mixing can shear DNA. Too much salt in the sample as well as rna although this runs much smaller then genomic dna. If your next stage is PCR then it really does not matter and sometimes degraded dna amplifies better then high molecular weight dna because it melts better. Alyhough yor dna is smeared it is probably still very high molecular weight compared with pcr product
DNA seems to b degraded. It depends upon the method you use for purification. Change the protocol and avoid contamination of DNases to get intact DNA.
Hii..
Do you have a gel pic with both DNA, before and after extraction..?? please upload.
And i have a doubt about your DNA, as you mentioned you have 4.16 ugml-1 DNA it is too low (4.16 ng per ul).
- Badges
- Science method