[vehuardo]

Still no one get this fragmentase to work?

Hi all,

seems like the NEBNext fragmentase isn´t the most popular enzyme in town.. Is there anyone in here who actually successfully use fragmentase on a routine basis?
I had planned to use it for prepping a very large number of microbial genomes. If it doest really work, or if I have to optimize for every sample, I should find another way. I don´t think there is a Covaris E220 available anywhere close and S220 will be a hassle, and expensive...

cheers

[vtosha]

We found no biases or difference between libraries after Covaris and fragmentase. If you have no possibility to use Covaris, fragmentase - best and cheapest variant. But I advise to prove length of fragments on each sample after treatment.

[vehuardo]

Thanks for your reply!

I actually prepped 8 M. tuberculosis illumina libraries yesterday, as a preliminary test. Fragmentation was carried out with fragmentase. After final amplification and clean-up, all 8 eight libraries have a relatively constant size distribution (230-300). I attached my crappy cell-phone image....

More variation in concentration. I suspect that AMPure beads dont give a consistent elution efficiency, as the balance between dry and over-dry is a fine one...?

Cheers

Vehuardo

[vehuardo]

BTW, gel image

Couldn´t upload: too big. I´ll try again later, have no image edit software on this computer...

[vtosha]

No need. I mean you should prove length just after treatment of fragmentase. I'm afraid, length of final libraries 250-300 too short for sequencing (length of insert 130-180). I recommend for bacterial libraries use insert 200-400 nt (350-550 final libraries). Try to use fragmentase with another time periods.

[kmkocot]

Of 3 tries with 15, 20, and 40 minutes on 8 diverse invertebrates (extracted with the Qiagen DNEasy kit) only one worked. The rest looked like unfragmented gDNA after even 40 minutes.

[vtosha]

May be secondary structure of DNA too difficult for fragmentase. If 40 minutes does not help, use physical methods of fragmentation.