Hi,
I'm working on preparing ATAC-seq libraries. After tagmention, the DNA were amplified very slowly, and it takes 15+ cycles to reach exponential phase of pcr amplification.
I measured the amount of DNA by Nanodrop. Even if there are more than 1 ng of DNA, it still takes 17~20 cycles to get enough DNA for sequencing.
It seems like pcr were inhibited somehow. I've read that if Tn5 transposase were not removed from DNA fragments, it will inhibit pcr amplification. But I've tried proteinase K digestion, SDS treatment, adding EDTA, purifying by Zumo column. But none of them worked.
So here is my problem: I got more than 1 ng of tagmented DNA, but they were amplified very slowly. Any opinions are greatly appreciated!
I'm working on preparing ATAC-seq libraries. After tagmention, the DNA were amplified very slowly, and it takes 15+ cycles to reach exponential phase of pcr amplification.
I measured the amount of DNA by Nanodrop. Even if there are more than 1 ng of DNA, it still takes 17~20 cycles to get enough DNA for sequencing.
It seems like pcr were inhibited somehow. I've read that if Tn5 transposase were not removed from DNA fragments, it will inhibit pcr amplification. But I've tried proteinase K digestion, SDS treatment, adding EDTA, purifying by Zumo column. But none of them worked.
So here is my problem: I got more than 1 ng of tagmented DNA, but they were amplified very slowly. Any opinions are greatly appreciated!
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