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-   -   Check transposon activity (http://seqanswers.com/forums/showthread.php?t=89530)

sobbewoscht 05-25-2019 09:31 AM

Check transposon activity
 
Hi all,

We are working with the Nextera Tagmentation Library Kit on 3 kb PCR products, to eventually pool libraries of 96 PCR reactions for a MiSeq run.

Tagmentation is done by plainly adding the enzyme + buffer to a dilution of the PCR reaction (no purification).

This generally works very well. However, for my last two library preps, my bioanalyzer traces ended up with a large peak at 3 kb (likely the original input), and nearly nothing in the 300-450 bp range.

This makes me believe that tagmentation was not effective.

Two potential culprits:

1) New PCR buffer (NEB Phusion HF Buffer) that includes detergent. Might not have been diluted enough and could have inhibited the transposon (final dilution of PCR buffer during tagmentation step was 1:25).


2) Loss of transposon activity. Kit technically expired six months ago; and it was accidentally thawed for several hours a few months ago. But we used it successfully after this accidental thaw.


I was thinking about an easy way to confirm that transposon activity is still there (without having immediate access to a bioanalyzer):

Purify PCR product > Tagmentation step according to Nextera protocol > Check whether the PCR band (ca. 3 kb) is gone (i.e., only smear or nothing detectable left).

Would that make sense to identify a loss in enzyme activity? Or would we not expect full tagmentation (i.e., complete loss of the 3 kb bnad) even for "fresh" enzyme.


Thanks!

nucacidhunter 05-25-2019 06:48 PM

I would suggest to do a reaction according to standard protocol from start to end and check the resulting library. Tagmented DNA will not migrate according to size becuase:
1- transposon will be engaged with DNA and slow its migration speed
2- presence of salts in the buffer


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