gDNA had smear band - problem in extraction human genomic DNA (Oct/27/2010 )
After I extracted gDNA from human cell line and determined by 1% agarose gel (new TBE), the smear band appeared.
We also found some smear band lower 0.5 kb. (Attached picture)
What is the problem ?
What is the small band lower 0.5 kb ? Isn't it RNA ?
It may be the result from old phonol/chloroform or RNase.
I notice that the old phonol/chloroform or RNase were used in this experiment.
It is probably RNA. Treat the DNA sample with RNAse and see if the smear goes away.
You mentioned old phonol/chloroform. What colour is it? Was 8 hydroxyquinoline added to the phenol-chloroform solution. 8 hydroxyquinoline turns the phenol-chloroform solution from clear and colourless to bright yellow.
If the phenol is red or brownish red, do not use it.
Alternative if the bright yellow has turned more like light brown do not use the solution.
In both instances the phenol has been oxidised and will damage your DNA prep.
I have noticed that samples in lane 3 and 5 have a long tail. This is not good as it indicates excessive DNA shearing. Avoid using the vortexer when preparing genomic DNA.
i'm not getting good yield of my g-DNA from h9c2 animal cell lines.and i'm not really sure if my phenol's fine or not.
as you mentioned above,if the bright yellow has turned like light brown,i shudn't use it.
i can't decide if my phenol's bright yellow or light brown!
any other way of deciding?