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  • #16
    Originally posted by ECO View Post
    That may be helpful...happen to remember the correlation of volume-shearing?
    No, it wasn't really mentioned. It was a just a quick remark he made while he was talking about process improvements for their illumina workflow. Just an off-the-cuff remark that it was more important that they had the tubes full than had the beads in there. They used beads until they found that it was only the volume contributed by the beads that was important, not the beads themselves. It may not be relevant, though, because they have their machine shop produce special glass tubes for use in their system (which is all robot-loaded!). It was Andrew Barry from the Broad that was talking about it, if you want to try to contact him about it.

    Scott.

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    • #17
      Hi,

      Can anyone please tell me where can I get information about the various factors affecting the size of fragmented DNA when using sonication and nebulization, that is how the size of the DNA fragment alter with the parameters.

      Thank you

      Comment


      • #18
        i use the sonication Kendra suggested and testing it by doing this (using the bioruptor UCD200):

        maintain water bath at 4C (if you have circluating bath it is easiest, or just add ice every 5 minutes as the energy will warm the small bath dramatically). i have found the shearing can be tested using TE vs the Illumina buffer (Neb) alone b/c the nice part is you can dry it down and look at shearing on gel to optimize, Neb has some salts that affect this. i put a sample in multiple 1.5 ml ep tubes and remove one every 10 minutes...200 ul volume, constant concentration (i used 25ng/ul), high setting, cycle for 30 sec sonication on/30 sec off...up to about 60 min. Run 1 ug of each on a gel (dry to 10 ul first) and find a timepoint that will meet your experimental objectives.

        after picking a timepoint with optimum fragment sizes using TE, try it with Neb, pretty similar sized smear except the uniformity (location and density at middle of the smear) is slightly different. with real samples i use TE for 40 minutes and get a smear from 100-450. The middle and darkest part of the smear is ~200-300, i cut this out and get hugely dense lawns w/ these libraries (~150-160k/tile using 1.4mm flowcell on GAII).

        Comment


        • #19
          Just wanted to make a quick comment on the effect of volume with Covaris. I work with Andy Barry, so he was probably referring to an experiment I did last year. Anyway, I found that the increased volume only helped with high energy settings meant to produce 100 bp fragments. I was using glass tubes from MicroLiter, which can hold about 300 ul. If you try to shear with high energy and a low volume, say 100 ul, you'll get a broad distribution and poor reproducibility. The guys at covaris told me that this happens because splashing creates an air pocket that deflects some of the acoustic energy. If you have beads in the solution, they will break the air pocket and you'll get nice tight reproducible shearing at 100 bp. Or as Andy mentioned, you can simply fill the tube, so there is no air to form any pockets. However, if you're trying to get 200bp-1.5 kb you can use smaller volumes with no beads and still get relatively tight reproducible shearing. Personally, I think Covaris is the way to go for small fragments (<1,5kb), and Hydroshear for larger. However, I am trying to develop Covaris methods that will give 4 kb fragments with distributions comparable to Hydroshear. If anyone's had success on that front please let me know.

          Comment


          • #20
            ECO

            Could you please update on your experience with HydroShear? Do you have the custom assembly as well as the standard? Any information would be great.

            Thanks!

            Comment


            • #21
              Hey there kwebb, sure thing.

              I have been using the hydroshear for a month or so with what I see as great success for fragmentation. My targets have been fragments in the 1-3kb range using bacterial DNA as the starting point, and I've found that the documented speed codes work quite well for honing in the desired range. An example is attached showing the effects of the four different speed codes. I'm only using the standard attachment but I believe others in the lab have tried the larger size shearing apparatus.

              The only downside is that the instrument is somewhat laborious to use when you have multiple samples (or high quantity of DNA), as not only do the shearing cycles take 10-20 minutes each (mostly walkaway), but the inter-sample cleaning routine is quite a bit of work (3-4x acid washes, 3-4x base neutralizations, 3-4x water rinses). They offer an automated valve which would reduce a lot of the annoying "turn the lever this way...purge...turn the lever that way...aspirate...repeat".

              I've had a couple broken syringes, one due to clogging and one due to what I think is a minor flaw in their programming for finding the "home" position for the syringe pump. Otherwise, it's pretty easy to replace the syringe and maintain. It's a pretty simple instrument.

              Genomic Solutions was very gracious in helping me get the machine up and running quickly, even sending me the Hamilton part number for the replacement syringe when they were out of stock.

              Hope that helps, I'll be happy to answer anything else I can.
              Attached Files

              Comment


              • #22
                maby, there are DNA betwwen 100-200bp,just you can see

                Comment


                • #23
                  Our Covaris just arrived, so I've got a bit of experimentation ahead of me. Can anyone share the paramaters they're using for ~200bp gDNA, or is the standard 20% duty, intensity 5, 200 cycles, 90s good enough?

                  cheers,

                  scott.

                  Comment


                  • #24
                    Originally posted by ZZ81 View Post
                    Just wanted to make a quick comment on the effect of volume with Covaris. I work with Andy Barry, so he was probably referring to an experiment I did last year. Anyway, I found that the increased volume only helped with high energy settings meant to produce 100 bp fragments. I was using glass tubes from MicroLiter, which can hold about 300 ul. If you try to shear with high energy and a low volume, say 100 ul, you'll get a broad distribution and poor reproducibility. The guys at covaris told me that this happens because splashing creates an air pocket that deflects some of the acoustic energy. If you have beads in the solution, they will break the air pocket and you'll get nice tight reproducible shearing at 100 bp. Or as Andy mentioned, you can simply fill the tube, so there is no air to form any pockets. However, if you're trying to get 200bp-1.5 kb you can use smaller volumes with no beads and still get relatively tight reproducible shearing. Personally, I think Covaris is the way to go for small fragments (<1,5kb), and Hydroshear for larger. However, I am trying to develop Covaris methods that will give 4 kb fragments with distributions comparable to Hydroshear. If anyone's had success on that front please let me know.

                    Hmm... the Covaris people just told me to try to get 50:50 solution:headspace in my samples! Maybe they weren't talking about DNA...

                    Comment


                    • #25
                      could you please explain what is the near target problem? txs

                      Comment


                      • #26
                        We are actually fragmenting via nebulization with satisfactory results applying 60psi for 4 minutes. Doiong this on several samples is not fun, so we shifted to NebNext Fragmentase, which also gives nice results.
                        We also tried Sonicman, but it never worked...

                        Comment


                        • #27
                          I would like to make a few comments in response to a few of the posts above which I hope will prove helpful:
                          1. The use of glycerol for DNA shearing using the Covaris instrument is not recommenced since glycerol absorbs the acoustic energy and heats up the sample during processing. The recovery of sheared samples containing glycerol, and at concentration, it quite low. Thermal biased shearing is also an issue when using glycerol.
                          2. Covaris also recommend not using glass beads for shearing DNA as the frictional heat of the beads will reduce sample recovery.
                          3. Covaris recommends using 120ul volume when processing samples using the MicroTubes. This will prevent splashing, and the formation of air-gaps which will cause an increase in the size distribution.
                          4. If the goal is to have a shearing of around 100bp, then a setting of 10%dc/5i/200cpb/960 seconds should be used. The sample should be in 120ul volume in TE or Qiagen buffer EB. I have posted a BioAnalyzer Trace.
                          5. Reducing the process time mentioned in 3, will generate fragments of around 130-150bp which is currently used by the new SOLiD protocol.
                          6. Covaris now does have validated protocols and consumables for generating 2kb, 3kb, and 5kb fragments. The distribution of fragments is as good, if not better than a Hydroshear. I have posted BioAnalyzer traces of 3kb, 3kb, and 5kb fragmentation using Covaris AFA.

                          Please let me know if I can provide any further information.

                          Thank you

                          Hamid
                          Attached Files

                          Comment


                          • #28
                            Hi Hamid,
                            Would you happen to have any information on the chemical status of the ends of DNA fragments produced using sonication? I detailed my understanding of an ancient paper on this subject in another thread:

                            Techniques and protocol discussions on sample preparation, library generation, methods and ideas


                            One comparison I would like to see for the various fragmentation methods would be the New England Biolabs-style "% of fragments could be religated after fragmentation". This could be with or without typical (T4poly/T4PNK) end repair.

                            Alas, even this would not address whether the various fragmentation techniques (or, indeed, DNA prep techniques) introduce chemical damage to DNA that cause most fragments to be not replicatable by a typical polymerase like Taq pol. But I can't think of a method that would address this issue.

                            --
                            Phillip

                            Comment


                            • #29
                              Does somebody else use NEB fragmentase for fragmentation? Is it gives some biases (practically) and if so what biases?
                              Last edited by vtosha; 02-21-2011, 06:46 AM.

                              Comment


                              • #30
                                Originally posted by ZZ81 View Post
                                Just wanted to make a quick comment on the effect of volume with Covaris. I work with Andy Barry, so he was probably referring to an experiment I did last year. Anyway, I found that the increased volume only helped with high energy settings meant to produce 100 bp fragments. I was using glass tubes from MicroLiter, which can hold about 300 ul. If you try to shear with high energy and a low volume, say 100 ul, you'll get a broad distribution and poor reproducibility. The guys at covaris told me that this happens because splashing creates an air pocket that deflects some of the acoustic energy. If you have beads in the solution, they will break the air pocket and you'll get nice tight reproducible shearing at 100 bp. Or as Andy mentioned, you can simply fill the tube, so there is no air to form any pockets. However, if you're trying to get 200bp-1.5 kb you can use smaller volumes with no beads and still get relatively tight reproducible shearing. Personally, I think Covaris is the way to go for small fragments (<1,5kb), and Hydroshear for larger. However, I am trying to develop Covaris methods that will give 4 kb fragments with distributions comparable to Hydroshear. If anyone's had success on that front please let me know.


                                Covaris consumable shears DNA to 6kbp - 20kbp fragments
                                g-TUBE, the latest innovation from Covaris, is a single-use consumable that enables scientists to shear genomic DNA into selected fragments sizes ranging from 6kbp to 20kbp. The only equipment needed is a compatible bench-top centrifuge and a pipette.

                                g-TUBE uses centrifugal force (the "g" in g-TUBE) to push the sample through a precisely manufactured orifice in the embedded ruby. This produces shearing forces in the sample that fragment the DNA. Fragment size is selected by adjusting the centrifuge rotor speed which alters the flow rate through the ruby and thus the shearing forces. Higher centrifugation RPM will produce shorter fragments and the protocol provide by Covaris contains the settings needed to select a fragment size between 6kbp and 20kbp.

                                Remarkably, the entire process takes only 3 minutes and up to 24 samples can be processed simultaneously.

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