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Frequently asked questions
- FAQ Bioruptor®
FAQ Bioruptor® | Tubes, attachments...
- Is there any recommendation concerning the volume of the samples in the tubes?
- Is it possible to sonicate 50 µl of sample in the 1.5 ml tube?
- Which kind of tubes is better for the sonication: made with “hard” or “soft” plastic?
- Which 0.5 ml tubes can be used for shearing of chromatin and genomic DNA?
- Can the O-rings for the 15 ml Adaptor (ref. P-10-A) be used for both orange (Corning) and blue (Falcon) capped tubes?
- If using Falcon tubes, which tubes are the best for use with the Bioruptor®?
- Concerning the 15 ml tubes, is it possible to use them without the reflecting bars?
- We are using the Diagenode 1.5 ml TPX tubes. They have worked well. Now suddenly they seem to be making white particles go into the solution and the liquid in the solution becomes very hydrophobic. We are using our Bioruptor on HIGH Power for 20 min - 40 min. Do you understand why this is suddenly happening?
- Which adaptors should be used for the 50 ml tubes from Greiner Bio-one (227261)?
FAQ Bioruptor® | Bioruptor use and care
- What are the critical points when using the Bioruptor?
- Which part of the Bioruptor is the most critical/fragile?
- Is it possible to sonicate continuously for a 10-15 minute time period without pause?
FAQ Bioruptor® | Chromatin shearing – Critical points
- a. Fixation step
- b. Cell lysis
- c. Shearing
- What types of cells are difficult to shear?
- What is the amount of cells per shearing trial to use?
- What is the key shearing buffer component?
- How long is chromatin shearing?
- What is the best cycle?
- What is the best volume/tube for shearing?
- What is the best temperature for shearing?
- How to maintain the low temperature during the shearing?
- How can I increase my sonication efficiency?
- d. Determination of the size of sheared chromatin
FAQ Bioruptor® | Other application
- Is it possible to shear DNA samples to single nucleotides using the Bioruptor® 200?
- Is it possible to sonicate DNA with the Bioruptor® and achieve the fragments between 2 kb and 5 kb?
- Does the Bioruptor® feat for shotgun library preparation?
- Could I use the Bioruptor® for cell lysis?
FAQ Bioruptor® | Tubes, attachments...
- Is there any recommendation concerning the volume of the samples in the tubes?
- Yes, for each kind of tube there is a minimal and maximal volume recommended as it is shown in the table attached below. For efficient shearing and reproducibility of the results.
| Description | Maximum volume per tube (or cup) | Minimum volume per tube (or cup) |
|---|---|---|
| 0.5 ml tube | 100 µl | 10 µl |
| 1.5 ml tube | 300 µl | 100 µl |
| 15 ml tube | 2 ml | 500 µl |
| 50 ml tube (Falcon or Corning) | 20 ml | 3 ml |
| 50 ml tube (Nalgene) | 8 ml | 1 ml |
- Is it possible to sonicate 50 µl of sample in the 1.5 ml tube?
- The minimal recommended volume for sonication in 1.5 ml tubes is 100 µl. It is better to respect it; otherwise your sample might disperse. Moreover, the volume smaller than recommended may introduce the variability between samples.
- Which kind of tubes is better for the sonication: made with “hard” or “soft” plastic?
-
The efficiency of sonication in “hard” plastic is much higher than in “soft plastic”; the transfer of ultrasonic waves is higher in the first type (“hard” plastic: polystyrene or polyethylene - crystal clear tube). The “soft” plastic (polypropylene, whiter aspect) can be also used, but the efficiency will be lower, so in case of problem with the efficiency the user should try the “hard” one.
There are also a special tubes made in plastic TPX with a better ultrasound transfer rate. The sonication is more efficient using these tubes. There are 1.5 ml and 15 ml TPX tubes available at Diagenode.
- Which 0.5 ml tubes can be used for shearing of chromatin and genomic DNA?
- We have successfully used the Sarstedt 0.5 ml microtubes (#72.699) or the Costar® (Corning USA) Rnase/Dnase-free microcentrifuge tubes (catalog #3206).
- Can the O-rings for the 15 ml Adaptor (ref. P-10-A) be used for both orange (Corning) and blue (Falcon) capped tubes?
- Yes. If you use another brand of tubes, please try the one which fits the best. The metallic bar should not touch the wall of the tube.
- If using Falcon tubes, which tubes are the best for use with the Bioruptor®?
- We recommend the BD FalconTM catalog# 352095.
- Concerning the 15 ml tubes, is it possible to use them without the reflecting bars?
-
No, the 15 ml as well as 50 ml tubes should be always used with the reflecting bar. This metallic bar in contact with the sample facilitates the transfer of the ultrasounds inside the tubes. The metallic bar is not a probe (no corrosion problems) but “reflects” the ultrasounds originated from the water bath and improves the sample sonication efficiency by a patented resonance system.
In case of 15 ml tubes the special aluminium ring should be used (to ensure an optimal position of the tube during sonication).
- We are using the Diagenode 1.5 ml TPX tubes. They have worked well. Now suddenly they seem to be making white particles go into the solution and the liquid in the solution becomes very hydrophobic. We are using our Bioruptor on HIGH Power for 20 min - 40 min. Do you understand why this is suddenly happening?
-
The advantage of TPX tubes is that they facilitate the sonication comparing to other tubes. In some cases, long sonication may cause plastic damage which will not interfere with the results. Simple centrifugation of the tubes will eliminate the issue of the particles.
If the user needs to sonicate for long time – par example 40 min, it is necessary to control the temperature of the water in the tank (changing the water or using the cooling system, making the pauses during the sonication to cool down the Bioruptor).
- Which adaptors should be used for the 50 ml tubes from Greiner Bio-one (227261)?
- "Universal adaptors for 50 ml tubes" x 3 + “Gear plate for 50 ml tubes” x1 = ref: UCD-pack 50; description: Universal adaptors for 50 ml tubes
FAQ Bioruptor® | Bioruptor use and care
- What are the critical points when using the Bioruptor?
-
- Control the temperature of the water in the tank – temperatures above 40°C can destroy the sonicator. The exchange of warm water with cold water or the use of the cooling system is critical. The Bioruptor® should not work overnight or during long time without any control as the high temperatures can cause the water to evaporate.
- Always assure water is available for the Bioruptor® to function
- Control the level of the water in the tank – if the level is not correct, the emitter of ultrasound can be destroyed. The tank should be always filled up to the blue line.
- Always use the aluminium rings with the adaptors for the 15 ml tubes to avoid damage to the tank.
- Empty the water tank by using the plastic pump. Do not tilt the water bath to pour out the water. The ultrasound emitter in the unit contains fragile mechanisms.
- Which part of the Bioruptor is the most critical/fragile?
- The water bath as the ultrasound emitter in the unit contains fragile mechanisms.
- Is it possible to sonicate continuously for a 10-15 minute time period without pause?
- Yes, it is possible. Nevertheless we suggest the pauses to cool down the ultrasound emission system. The ultrasound emission system located below the water bath will heat up and overtime may decrease the life of the instrument.
FAQ Bioruptor® | Chromatin shearing – Critical points
a. Fixation step
- What is the formaldehyde final concentration to use?
-
1%.
Is it important to use only fresh and good quality formaldehyde. That is why we recommend to use the small bottles of formaldehyde. ChIP grade formaldehyde is available at Diagenode.
- How long is the fixation step?
-
Fix for 10 minutes. It is possible to fix for as little as 5 minutes (depending on your protein of interest for subsequent ChIP assay). Use glycin to quench the fixation and wash well the fixed cells with cold PBS to eliminate all traces of formaldehyde.
Too long fixation can provoke over-fixation, what will decrease dramatically the efficiency of sonication.
- What is the temperature to use for fixation?
- Fixations at RT and at 37°C are possible. Note that at 37°C the reaction is faster than at RT.
- How to stop the fixation?
- Diagenode recommend to stop the fixation by adding 125 mM glycin for 5min, followed by 2 washes with PBS. This method allows to eliminate all trace of formaldehyde.
b. Cell lysis
- Is cell disruption complete?
- Do not use too many cells in the cell lysis buffer. Lyse about 5x 10e6 cells/ 1 ml.
c. Shearing
- What types of cells are difficult to shear?
- Cells growing in a suspension culture as fro example blood-derived cells (monocytes, macrophages, lymphocytes) are known to be more difficult to shear.
- What is the amount of cells per shearing trial to use?
-
1x 10e6 - 10x 10e6cells/ 300 µl
3x 106 - 30x 106 cells/ 1 ml
Too high cell density will decrease the efficiency of sonication.
- What is the key shearing buffer component?
- The shearing buffer should contain the detergent. Quality and quantity of detergent is very important. Par example Diagenode recommend the SDS, its concentration in the shearing buffer should be 0.75-1%.
- How long is chromatin shearing?
-
The time of shearing depend on many factors, like: cell type, cell density, volume of the sample in the tube and amount of the tubes during the sonication, fixation time, concentration of detergent in the shearing buffer, etc. So it is important to optimise the sonication conditions by each Bioruptor® user.
Generally the shear time is 15 minutes. It is possible to shear for as little as 5 minutes and up to 30 minutes maximum (for longer fixation period: a longer shearing is needed).
- What is the best cycle?
- 30 seconds «ON» + 30 seconds «OFF».
- What is the best volume/tube for shearing?
-
1.5 ml per 15 ml tube
200 µl per 1.5 ml tube
Do not use a too big sample volume.
- What is the best temperature for shearing?
- 4°C. Keep it cool all the time.
- How to maintain the low temperature during the shearing?
-
There are 3 possibilities:
1. Automatic temperature control
A refrigerated circulation bath can be used to guarantee the automatic temperature control of the water bath during the whole sonication process.2. Manual temperature control
Before starting the first round of sonication a “pre-cooling” of the Bioruptor®’s tank with crushed ice during 15 minutes is advised. This will allowed to avoid too quick water heating due to thermal inertia (the tank and the ultrasound generating elements are generally stored at room temperature). Then cold water should be added to the crushed ice in the waterbath up to the indicated level (sticker).We suggest you to keep a stock of water at 4°C (fridge or cold room). The crushed ice floating in the water should not exceed 0.5cm and the total water level (water and ice) should be exactly at the indicated water level.
At the end of a typical sonication time (10 minutes, cycles of 30 seconds “ON” & 30 seconds “OFF”), the temperature in the water bath should stay below 10°C.
3. The permanent installation of the Bioruptor® in a cold room is possible, although not sufficient to avoid the temperature increase due to sonication. This location would only suppress the “pre-cooling” step described above.
- How can I increase my sonication efficiency?
-
A few points to follow for optimizing shearing efficiency:
- Do not perform excessive fixing of the cells.
- Make sure the FA buffer used is fresh – quality of this buffer is critical.
- Make sure you stop the fixation reaction with glycine and that you wash the cells to eliminate all traces of FA.
- Performing the lysis step, do not use to many cells in the lysis buffer.
- Use the detergent in your sonication buffer (par example 0.75-1% SDS).
- Use the tubes made with “hard” plastic.
- Respect the volume of the sample in the tube during the sonciation.
- Try to sonicate the samples during different times and choose the best option.
- Try to sonicate the sample with different concentration to check which gives the best results.
d. Determination of the size of sheared chromatin
- What kind of gel should be use for checking of shearing efficiency?
- The DNA disruption after shearing should be tested on 1%-1,8% agarose gel. The gel should be freshly prepared. For accurate size determination of the DNA fragments, the reversion of the cross-linking and DNA precipitation after phenol/chloroform extraction is advised.
- Is it necessary to reverse the cross-linking of the chromatin before analysing it on the gel?
- Estimation of the size of chromatin fragments without the reversion of cross-linking is not too precise. The presence of the proteins which migrates with the samples on the gel causes that the migration is longer and less precise. However if the conditions of sonication are well optimised, it is possible to avoid the long phase of the cross-linking reversion and purification; instead of that it is possible to perform only general control to check , if there is no major problem with the shearing.
- How much of sheared DNA should be loaded on the gel?
-
Do not load more than 5 µg/lane.
The migration of large quantities of DNA on agarose gel can lead to poor quality pictures which do not reflect the real DNA fragmentation. Do not load too much on a gel. Also we recommend to treat the sample with RNase. The minimum amount that can be visualized in agarose gel corresponds to 100 000 cells.
- Which running buffer (which concentration) should be used?
- 1X TAE or TBE is preferred to 0.5X TAE which can lead to smears on gel. The buffer should be freshly prepared.
FAQ Bioruptor® | Other application
- Is it possible to shear DNA samples to single nucleotides using the Bioruptor® 200?
- It is not possible to get single nucleotides with Bioruptor®. The lower limit is about 100-200 bp.
- Is it possible to sonicate DNA with the Bioruptor® and achieve the fragments between 2 kb and 5 kb?
- Diagenode has not tested protocols to get DNA of 2-5 kb. However, we have conducted time course experiments on DNA shearing which have shown that after 1 minute at low power, we achieve an average DNA size of 800-1000 bp. Thus, it is possible to get larger fragments by reducing sonication time. We recommend your own optimization for such applications.
- Does the Bioruptor® feat for shotgun library preparation?
- Yes, Diagenode’s Bioruptor® Sonicator is recommended for next generation sequencing over nebulization and other sonication devices – this information is published in a recent Nature Protocols publication: Hodges et al, 2009.
- Could I use the Bioruptor® for cell lysis?
- Yes, the Bioruptor® is widely used for cell lysis using bacteria and other types of cells, for protein extraction, etc. More information about different application you can find in the section “Protocols”.