When I run a gel on the extracted DNA from my samples I always notice smear with varying degree of strength what do they mean?
a picture is attached to clear out my point
I store the samples at-20 for 4 months before beginning my DNA extraction can this storage condition cause degradation of DNA ?
I used 0.7% agarose gel and run on 200-220 volt for 20 min
I used iagen blood and tissue spin-column protocol
Hi Asmaa Nasr ,
I agree with Rommel, you have samples with high-grade degradation, do you have more information about your DNA isolation protocol? Is this performed with some commercial kit or in-house protocol?
Regards,
Greetings,
There are could be several reasons to why your DNA is degraded. Your kit is Qiagen DNeasy Blood and Tissue Kit, and I assume you used the protocols for the insect. It could be mechanical shearing or DNase contamination and etc.. You mentioned storing your samples in -20, it could be one of the reason, if you frequently and repeatedly thaw and freeze your samples.
Anyway, here are some questions:
1) How did you homogenize your samples?
2) Did you performed your extraction in low temperature (on ice)? I don't remember the protocols from the kit mentioned about performing at low temperature.
3) What are the A260/A280 readings and DNA concentration?
I cut the sample using scalpel blades into small pieces
The 260/280 were from 1.5: 2 in all my samples concentrations were from 50 to 200 or 300 but there are samples were concrntrations are less than 50 and high than 1500
No i didnot perform extraction on ice
I dont remove my samples from -20 except for extraction
But the thermometer some times usually read -15 and -13
DNA not affected by storage at -20C but, repeat freezing thawing may degrade the DNA sample.
Greetings,
The DNA degradation might be due to the nuclease released during homogenization. Might I suggest preparing 1mL 1X PBS + 2uL EDTA, and proceed with the kit's protocol. During homogenization, do perform it in an ice box to lower the nuclease activity.
Your DNA is highly degradated, can you give us more information about your protocol? I suggest this paper for you Article An Alternative and Rapid Method for the Extraction of Nuclei...
I do not usually make a gel to test the integrity of the DNA samples. Instead, try to dilute your DNA samples may be 1:20 and make PCR directly using your primers, see the result.
Best wishes
Do you have information on your extraction procedure? Shearing can be the result of being too rigorous with bead beating/centrifuging for example. Or not working fast enough and on ice. When I worked with fish gut content - these degraded very very fast due to the nature of the material. You can try optimise your extraction procedure to reduce the amount of shearing. The integrity of the DNA used in downstream processes is important.
My samples are ticks
I used minicing of tissue using scalpels with overnight incubation at lysis buffers
Could it be the cause for this degradation?
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