Hi!
I am using the XT Nextera kit with environmental samples. DNA was extracted and purified by phenol/chlo and column (to remove EDTA).
When I used the recommended DNA final quantity (1ng) I didn't get anything. If I used less than that, it was worst.
I have to increase the DNA amount a little bit (1,2ng final) and add one cycle to the PCR to get a pic with the correct size (around 1000b) for the MiSeq run. But I still have a low yield.
I purified the library with AmPure beats 0.5x.
Any advice to increase the yield? Maybe a way to remove degraded DNA before tagmentation? Is it possible to make a home-made PCR instead of the XT polymerase?
Thanks!
I am using the XT Nextera kit with environmental samples. DNA was extracted and purified by phenol/chlo and column (to remove EDTA).
When I used the recommended DNA final quantity (1ng) I didn't get anything. If I used less than that, it was worst.
I have to increase the DNA amount a little bit (1,2ng final) and add one cycle to the PCR to get a pic with the correct size (around 1000b) for the MiSeq run. But I still have a low yield.
I purified the library with AmPure beats 0.5x.
Any advice to increase the yield? Maybe a way to remove degraded DNA before tagmentation? Is it possible to make a home-made PCR instead of the XT polymerase?
Thanks!
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