Bioruptor protocols for Truseq DNA sample prep

[HICORE]

Hello,

I am getting ready to prepare some libraries using the Truseq DNA sample prep kit using a Bioruptor for fragmentation. Before starting the actual samples (150,000 bp), I am running a test sonication on some Ecoli gDNA.

I ran my test samples at 15" on 90" off for 9 cycles. According to Diagenode this should approximately give a fragment size of 350 bp. I ran the samples on a Bioanalyzer DNA 1000 chip afterwards to size the fragments. It wasn't even close to 350 bp! Here is what I got:

Sample 1: 100 ul in EB at 20 ng/ul. Average size: 870 bp!

Samples 2: 52.5 ul in EB at 20 ng/ul (as the protocol asks for). DNA 1000 didn't pick up anything. Hmmm...

Therefore I am wondering what fellow sequencers run there Truseq DNA sample prep samples at on the Bioruptor?

I would be thankful for any input!

[aarodriguezm]

Hi Hicore
Is a nice try!!!
First you have to be sure the size you want to achive, what is your goal?
For a normal illumina 100 bp HiSeq the best Size is about 200 bp if you want to do a 2x 100 bp lane. If this is your case i will recomend you to do at least 20 cycles 30 s on 30 off.
You can also test it first with E.coli DNA first and run a chip.
Have fun

[HICORE]

Bioruptor protocols

Thank you aarodriguezm!

Do you find that sonicating samples of different lengths require different protocols even if they are the same concentration?

[aarodriguezm]

Hi again
In protocol you will find that the best amount of DNA is about 100 ng and the best volume to do it is 100 µl. My experience is that the lenght, concentration, volume, and temperature can be definitive. But as i said to you, you can run several tests to calibrate your best protocol for your sample. Each sample might be different even with the same concentration, same lenght and in the same protocol, but the differences are not so high.

[protist]

Hi, we've done many fragmentations with our Biorupter and have found the best parameters are 30sec ON/30sec OFF on high power. Make sure the bath is filled with water-ice mix. I usually do 15 min sonication and change water ice mix and sonicate for a further 15 min with the same power and time settings. Total volume is 200 uL and DNA is diluted with EB. Depending on the organism - we have used for many different euk and bacterial species - a total sonication time of 25-35 mins is generally sufficient to push fragments to 200-300 nt range. We generally sonicate anywhere from 1-5 ug DNA at a time.

[dalib]

Hello,

Before comparing protocols it is important to make sure you are referring to the same Bioruptor model. Diagenode offers different systems and the procedure to obtain a specific fragment size may vary between models. Here you can find the latest protocols for the different Bioruptors: http://www.diagenode.com/en/support/protocols.php

In general terms for DNA shearing Diagenode recommends using 100ul at 10ng/ul in 0.5-0.65ml tubes. Different volumes or concentration will require further optimization. For an efficient, reproducible and tight size distribution it is important to use the correct plastic type and the tubes from Diagenode offers the best performance (http://www.diagenode.com/en/catalog/...s-56/tubes-58/).

As mentioned by protist, it is very important to maintain constant the water temperature. Best results can be achieved by connecting the Bioruptor to a water cooler system (http://www.diagenode.com/en/catalog/...bioruptor--362).

Daniel