Bioruptor vs Hydroshear

[EMONGS]

Roche 454 libraries are designed for the Hydroshear. I have read various threads and it looks like the Covaris is much better than the Hydroshear. Since the Bioruptor works just as well as the Covaris, will it be safe to design parameters on the Bioruptor for 454 PE libraries?

If so, has anyone already tested different parameters on the bioruptor for various fragment sizes for the 454 PE libraries 3,8 and 20 kb?

[GW_OK]

The covaris is better than the hydroshear for <1kbp fragments. Beyond that the hydroshear is better, in my opinion.

[Hamid]

Dear EMONGS,

Covaris is the only technology that allows for highly reproducible shearing of DNA in the size range of 100bp to 5000bp.
Please take a look at the attached shearing data showing the reproducibility, concentration flexibility without the need for optimization, and validated protocols for a wide range of fragment sizes.

Thank you

Hamid

[EMONGS]

Well I am stuck with the bioruptor for some time and now I need to shear a tight product of 150-200 bp. Any insight/ protocols? Thank you in advance.

[Hamid]

Hi Emongs,

Why don't you email me your contact information. I can see if we could setup a demo for you next time we are in your area. We do have a sales manager in Knoxville, and he does travel to Alabama often for demos.

Thank you

hamid

[Liting]

Hi Emongs,

Why don't you email me your contact information. I can see if we could setup a demo for you next time we are in your area. We do have a sales manager in Knoxville, and he does travel to Alabama often for demos.

Thank you

hamid

hello，Hamid.We also use the bioruptor to shear the genomic DNA.We want a tight product of 200-400bp.Can you give me some advice or protocal?The instrument in our lab is diogenode UCD 600.
Thank you very much!

[pmiguel]

hello，Hamid.We also use the bioruptor to shear the genomic DNA.We want a tight product of 200-400bp.Can you give me some advice or protocal?The instrument in our lab is diogenode UCD 600.
Thank you very much!

Hi Liting,

Hamid works for Covaris.

Some of the others here might have a biorupter and have a protocol for you, though. (We have a Covaris.)

--
Phillip

[Liting]

Thanks a lot.

[madseq]

Hi Liting,

we´ve been setting up bioruptor conditions for our exome experiments.

Firstly, we have noticed that concentration of the sample makes a lot of a difference in the final fragment size. We found some information at diagenode´s site recommending to use Low power only (although I´ve just checked and they now have an updated information for HIGH power). So we did a time-course with 300 ng and low power. The water bath was set at 4ºC and the sample was diluted in a 100 ul final volume of TE.

For 200-400 bp I would recommend using 40 min if you start with 300 ng. However, when we switched to 1,5 ug I wouldn´t recommend less than 50 min.


We have sometimes found that not all the tubes reach the desired length, so to make sure we get the right fragment length we better do 50min at low power and then 5 minutes at medium.


Also, we have tried sonication in 2 different bioruptors. In one of them, the water temperature was maintained by an automatic pump. In the other one, the bioruptor was located in a 4ºC chamber and from time to time it was necessary to add some ice to the water. I don´t know if the ice disturbs the waves or something, but this latter configuration makes the sample not to fragment efficiently.

You can find a poster with the updated information here:

http://www.diagenode.com/en/topics/sonication/sonication.php

Hope this helps.

[Liting]

Hi Madseq,
Thank you very much.We have just noticed that the concentration of the sample makes a lot of a difference in the final fragment size.We have tried different conditions.The bioruptor is diagenode UCD-600.It has a cooler system.And right now for 200-400bp we use 30''on/30''off and 4cycles at high power with 2ug DNA.It's different from your condition.Pehapes we use the different instrument.Thank you again!

[dr.m.umer]

Hi
I have been using UCD-300 Bioruptor. I need fragment sizes between 100-250 bp (for solid). I do not seem to find an optimal protocol for this. I have used various quantities of starting DNA and fragment size does not seem to change a lot. Attached is a picture of optimization experiment. 12ug of total DNA was used in 450ul T.E. The conditions were, Low power, 30s on/30s off. Bath temperature maintained between 4 and 8 C. Approx 100ng DNA was removed after every 10 cycles and run on 2% agarose. the marker is 100bp. although the major chunk is still between the rquired range after about 70 cycles, I am still not satisfied with the results. Can someone suggest me a better protocol, or should I shift to Covaris (we have that too)

[ETHANol]

It is a no brainer. Shift to Covaris if you have it. Just make sure you use the correct tubes and follow their protocol!!!!

[dr.m.umer]

that is always an option, but i was just wondering if bioruptor can still be used. anyway, let me try covaris. one other small question in my mind. why do we purify after sonication, i think to remove the single stranded DNA. say if i have to do Methylated DNA IP between lib prep and sonication (which actually needs DNA to be single stranded), so would it make any difference if i purify after sonication or not.
may be this wrong thread for the question, but still if someone can answer, it would be helpful

[ETHANol]

one other small question in my mind. why do we purify after sonication, i think to remove the single stranded DNA. say if i have to do Methylated DNA IP between lib prep and sonication (which actually needs DNA to be single stranded), so would it make any difference if i purify after sonication or not.

I wondering the exact thing. My wild guess is that someone along time ago did a purification, it worked and it was enough of an inconvenience for anyone to change it.

Reasons to use the Bioruptor for fragmentation of your DNA for MeDIP: 1) you have one in your lab, 2) if you have to buy one it is much cheaper then the Covaris.

Reasons to use the Covaris for fragmentation of your DNA for MeDIP: 1) the fragmentation is highly reproducible, 2) fragmentation is not sensitive to concentration as long as you stay below the limits, 3) it works really well - nice tight distribution, 4) isothermic results in less bias and less denaturation.

Since you have the Covaris, use that.

Hamid, are you there: do you have conditions for shearing larger quantities of DNA in 12x12 tubes? The Covaris website only has conditions for microTubes. I gave it a try in 12x12 tubes using the settings for the microTubes and I got good shearing (200-300bp) with 6 min.

http://www.covarisinc.com/pdf/pn_400056.pdf

I'll also note with the Covaris the tubes always have to be completely full. 12x12 tubes take a little more then 1 ml.

[dr.m.umer]

I wondering the exact thing. My wild guess is that someone along time ago did a purification, it worked and it was enough of an inconvenience for anyone to change it.

I think you are right, why should I waste my time and DNA and Ab on this thing .....
I usually do not do purification, and my MeDIP seems to be working, but not very good (lot of high methylated regions in unbound fraction). I was just wondering if it has anything to do with purification (my mind can't accept though because I do not see any logic, may be if someone can come up with some point I do not know/understand)