I ran gel electrophoresis to see if my samples have chromosomal DNA in them (for later PCR usage). When I saw the results I didn't know how to interpret them. I understand the first line is the chromosomal DNA but what are the lower two lines?
Some of my samples before DNA extraction were frozen for some days in -20 C, 1x PBS. From other samples I extracted DNA immediately after collecting cells.
Those two lines are not visible from the samples I froze before extracting DNA...
DNA Ladder sizes: 10000 - 1500 (MassRuler, Reverse).
I will appreciate any help!
Thank you for your answer!
The marker is the right one. This why I excluded those lines to be RNA.
http://www.thermoscientificbio.com/uploadedImages/Products/DNA_Electrophoresis/DNA_Ladders/sm124_fam.jpg
Can it be that RNA subunits tend to stick together with each other?
I used Phenol/Chloroform extraction but by mistake I used acidic phenol, therefore the DNA is not that pure. However, I need this material only for detection if all the viruses from my stocks are alive and able to infect cells. Later I will need more purified material.
And if it is RNA, then why cells which were frozen before DNA extraction don't have it anymore?
This DNA you extracted, from which cell type?
Since I've read you work with adenovirus, may it be a cell line transduced with a viral vector? or transfected with a plasmid? Then, those lines could be two forms of the vector.
I agree with Andrei Nikonov
The fragments in the ladder don't look like the one you specify. The frozen samples lack the RNA because it is less stable than the genomic DNA and degrades with freeze/thaw (and probably just with time).
I have never found these ribosomal RNAs to interfere with downstream PCR, so if I were you I would ignore them. They will degrade very quickly during thermal cycling in any case.
cheers
Rob
I do not know it may be. But if I'm sure that's not RNA.
More information is needed. With that cells you have made the DNA extraction?.
Hi
You may add RNase A, to a final concentration of 20µg/ml to the samples and incubate them at 37 degrees for 30 min. In this way the RNAs would degrade. But if you want to use the DNA samples for PCR, no needs to treatment. you don't have any problem because the elevated temperatures of PCR can degrade RNAs.
God speed you
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA