- I have isolated genomic DNA from Klebsiella pneumoniae using promega wizard bacterial genomic DNA extraction kit. I have followed the protocol instructions. But I can see smears in my gel and small bands below from lane 1 to 7 (picture attached below). The samples were treated with 3ul Rnase soln at 37c and kept for 2hrs. Then treated with 2.5ul proteinasek (20mg/ml) and kept at 55°C for 1hr. then added protein precipitation solution and centrifuged at max speed. the soup was taken and mixed with 600ul isopropanol and centrifuged. the pellet was taken and washed with (chilled)70% ethanol and again centrifuged. Similarly when i have not treated the samples with proteinasek, there are no small bands below but still there is smear from lane 10 to 17. The concentration of the samples and 260/280 ratios are attached in the gel picture. Why there are smears in my gel and the small bands below? please give me suggestions how can i improve the quality of my DNA for nanopore sequencing.
Sanjoy Biswas
There are several cause for this smear . One- May be your elution buffer is not suitable. Two -Due to large genomic size DNA breaks take place. Try to isolate them in conventional method with some extra care . May be it will help you.
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