I would like to know from your experience where the mistake is
Experimental steps include the use of sodium salt calf thumus and was diluted in TE buffer size 50 ml and at a concentration of 2 mg / ml l . After dissolving DNA for 24 hours at 4 ° C was exposed to eletctron beam 6 Mev from elekta preise linac (dose = 80 Gy and dose rate = 5 G / min, field size 20 * 20)
Then 50 mL DNA was divided into two glass petri dishes and a depth of each DNA solution in petri dish was ~ 1.6 cm and 1.5 cm water equivalent material was placed directly above the two plates without placing the cap petri dish. Gel eletrophorsis were then performed after a week of exposure and the sample in that period was reserved for the refrigerator at 4 ° C
do you expect to see visible degradation?. If one of those samples is a control then it is already quite degraded so any degradation of other samples will simply move already degraded dna a little lower down the gel but looking very like the control. Ionising radiation will cause single stranded nicks but dna is very long and double stranded nicking causing breakages will be quite rare. I think that you need a more sensitive technique than agarose gel to measure degradation. Possibly some of the bright signal in the well might be very large dna but could also be protein like histones.
Thanks a lot for your answer.
The first two wells contain the control sample.
But how there are histones! I purchased a DNA package in the form of lyophilized fibers. Also, the purity ratio was good when calculating OD260 / OD280 = 1.9
Please let me know examples of sensitive characterization techniques to measure DNA degradation
What DNA is it ? Is it just the genomic DNA ? or any particular size/amplicon ? On what basis you are expecting degradation ?
- If you are using genomics DNA, you should reconsider to modified it to fragment based analysis. Since the fragmentation in genomic DNA itself is very much dependent on the DNA extraction method, you could not really access the level of fragmentation in genomic DNA. As can be seen in gel, all samples were a smear, which is due to the genomic DNA itself.
- 0.7 % is definitely not OK to see the differences in size. Use 2% or more.
- But, gel electrophoresis itself is not that technique, if one is not experienced in doing that. May be you can use bioanalyzer, which will give the ready made results. PAGE could be another option, but complicated.
I think that to get detail either HRM analysis of small pcr fragments to get an estimate of increased mutation in small fragments and extrapolate to longer dna or something like droplet digital pcr
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