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-   -   Nextera XT tagmentation problem (http://seqanswers.com/forums/showthread.php?t=66996)

nao 03-18-2016 05:10 PM

Nextera XT tagmentation problem
 
Hello all,
I need some help with Nextera XT library prep from bacterial genome.
DNA were extracted using QIAmp kit with QIAcube HT robot and measured the DNA by absorbance and fluorescence.
I am following the Illumina protocol strictly and checking the library after PCR cleaning step to decide to move forward or start over the tagmentation & PCR.

I had few samples to start over this time, but I am now stuck with tagmentation step for few days.
I suspected inhibitor contamination or wrong DNA concentration, so cleaned the DNA with Zymo gDNA cleaning kit and eluted with water to avoid EDTA. DNA was measured by absorbance and fluorescence again to make sure the concentration. I confirmed the genomic DNA by running on QIAxcel.

Brand new TD buffer and ATM buffer was used. After tagmentation (55 C, 5min), no tagmentation was going on confirming by QIAxcel.

Does anyone had same experience? The DNA were all extracted under same condition at the same time, so why does this difference happens?:confused:

In previous thread, it was recommended to clean with Mo-bio kit, so I will try that too.

Any help or suggestions will be appreciated! I am now thinking to re-extract the DNA.

nucacidhunter 03-18-2016 08:51 PM

Possible causes:
1- Input DNA
2- Reagent issues
3- Reaction set up practices
4- Thermocycler issues (can check the temperature with a probe or a thermometer)

To narrow down the cause I would set up reactions using another DNA that has worked fine before or a clean commercial DNA with the same reagents that were used for failed reactions. It will be useful to post electropherogram of sample before and after failed tagmentation.

nao 03-19-2016 10:35 PM

Hi nucacidhunter,

Thank you for your suggestions. I didn't think about the thermocycler, but definitely will check that or run with other machine. I will also try with other working DNA to see how it goes.

I prep 32 different DNA at a time and there are always some samples that don't go well. Usually it was some pipetting errors like that so when I repeat it from beginning, the final libraries look fine. This time I can't even pass the tagmentation step, which is very strange.

I don't have electropherogram now, but will post once I save them.

adam.geber 03-21-2016 03:11 PM

Nextera XT requires input DNA of fairly low concentration (0.2ng/uL). What platforms are you using to measure your samples' concentration? Are you using QIAxcel readings to make your dilutions before tagmentation?

As nucacidhunter mentioned, an electropherogram will be quite helpful. When you say that there is no tagmentation occurring do you mean that the traces from before and after look identical?


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