Nextera XT low yield

[nicoT]

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!

[Simone78]

Hi,
difficult to say why you got a low yield without seeing the whole protocol.
If the degraded DNA is leftover in the solution it will be tagmented, regardless of its length. Well, almost. Adey et al. (Genome Biology 2010) report that the smallest fragment that can be tagmented is ca. 40 bp. In our recent paper (Picelli et al., Genome Res 2014) we also saw that anything <100 bp is tagmented.
We are using KAPA HiFi non-HS kit for the PCR. Please take a look at the paper for the details.
I believe that (almost) any non-HS kit will work as long as you do a bit of optimization!
The XT Polymerase is just Phusion Polymerase, by the way. KAPA performs better, especially for GC-rich fragments.

you are concerned about adding a few extra PCR cycles but it shouldn´t make a big difference in the final quality of data. However, if you end up with a low-complexity library you need to solve the problem with the yield (maybe you are preferentially amplifying some fragments and losing others).

I hope this helps, good luck!
/Simone

[darwin142]

0.5 XP beads may be a little low, I would try at 0.85.