I am trying to amplify a DNA fragment of about 500 bp. The control which is the DNA extracted from the last cell sample. This DNA sample allows me to amplify the region, which proves that the primers and PCR conditions worked, but I used the same PCR materials and conditions to amplify the same fragment from the new DNA extracted from the new cell sample, it did not work. The only difference between these two cell samples was that the new sample got more than 50% cell death bc they lifted off the plate that I could not even wash the cells before harvesting, but I still harvested them anyway; and the old cell sample did not have any cell death because I washed off all the cell death before harvesting. The DNA extraction from both samples were at 50-100 ng/uL, so I don't understand that could be wrong with the genomic DNA that prevents the PCR from working normally. Please help!
yes, cell death will affect genomic DNA.
DNA will degrade. To test if DNA is degraded you can run some on a gel
~1% agarose you need to use 1-2 ug so you can see the band or smear
Good DNA should band near the top with only a faint smear, degraded DNA will smear as it is broken into small fragments
you should run a DNA ladder to get a size for the fragments
Hello, Kien,
I agree to Ingo's answer above. Cell death will definitely affect genomic DNA. The apoptotic process includes DNA degradation and in order to ensure successful amplification of your target sequence, you must be able to secure fresh DNA samples without contamination of PCR inhibitors. Run the DNA samples to check on their integrity.
You may refer to the following material on PCR inhibitors: https://www.promega.es/-/media/files/resources/profiles-in-dna/1001/an-introduction-to-pcr-inhibitors.pdf?la=es-es
Take note of steps where possible inhibitor is introduced.
Hope this helps.
Best,
Arizado
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