gDNA fragmentation NEBNext™ dsDNA Fragmentase™

[jgibbons1]

Hi all,
Was wondering if anyone has used New England Biolabs NEBNext™ dsDNA Fragmentase™ yet? I have been having some problems with sonication and was hoping to move to a more standardized method. Their assay looks good but I've found no publications yet that have used these enzymes. Anyone have any experience?
Thanks,
John

[gogreen]

I used the enzyme for just testing the fragmentation and was pretty impressed with the fragment size on the gel. It was a focussed smear at 100-600 bp. But when I ran the samples on the bioanalyzer after 2 days, I see a large smear ranging from 30 bp to 2000 bp peaking around 1.5 kb which does not correlate with my gel results!! I'm trying to figure this out by testing some more samples and will update this.

[jgibbons1]

Thanks for the response and comments. I will be testing this out on Monday and will let you know how everything works for me...Keep me updated on what you figure out though.

[waynes_world]

I used the enzyme for just testing the fragmentation and was pretty impressed with the fragment size on the gel. It was a focussed smear at 100-600 bp. But when I ran the samples on the bioanalyzer after 2 days, I see a large smear ranging from 30 bp to 2000 bp peaking around 1.5 kb which does not correlate with my gel results!! I'm trying to figure this out by testing some more samples and will update this.

we are also interested in using the fragmentase for sizing <200bp. did you purify the rxn once the initial run was complete to remove enzyme?

[peromhc]

Fragmentase.. where to begin. We used it for 3 gDNA library preps, 2 lizards and a mammal. The mammal genome fragmented sucessfully- 20 minutes, but I still had a lot of large fragments, my mean size was ~1kb based on bioanalyzer.. I ended up using the bioruptor on this samples after Fragmentase to get it smaller.

One of the lizard genomes did not digest- had to biorupt... maybe it was GC content or contamination, but these were all QIAGEN extracted and had good numbers via nanodrop.. We digected for as much as 2 hours!

The other lizard was somewhere in teh middle, digested, but not that good (i.e. large fragments).. Needed to use the bioruptor...

So, if you want to use fragmentase, plan on doing a lot of experimentation to get it right, and even then you might need another alternative method..

[jgibbons1]

Hi all,
I've been playing around with NEB fragmentase for the last week and have been running into a few problems. I was wondering if others have experienced these issues as well.

(1) I've tried a few assays including amount of starting gDNA (2ug and 5ug) and incubation time (10, 15, 20, 25, 30, 45, 60 minutes). Each time I run a check gel, the high molecular weight gDNA band is gone, but I do not have a smear. I was thinking that I was just oblivious and not executing the protocol correctly, so I called NEB and they sent me HeLa cell gDNA (one of the examples in their product pamphlet) to run as a control. I ran the HeLa sample and my 2 samples in one run all with final gDNA concentrations of 50ng/ul for a total of 5ug and the HeLa gDNA fragmented perfectly, but again, my samples no longer had the gDNA band BUT had no apparent smear (have stained with EtBr, Sybr green and GelStar).

(2) I've performed the same assay with different gDNA extraction methods including CTAB, Qiagen and Phenol Chloroform. Each time I am getting the same exact thing: degradation gDNA band but not smear. I was thinking that protein contamination could possibly inhibit the fragmentase but really and not sure what is going on and am becoming increasingly more frustrated.

On a side note...I tried using an older sonicator earlier (which is why I tried NEB fragmentase) and found the same exact pattern. Degradation of the gDNA band but no smear. This is leading me to believe that maybe my quantification is not accurate. I am using a nanodrop for initial gDNA quantification. I spec'ed the HeLa DNA and it spot on to what NEB said the concentration was (200ng/ul). Also, the phenol chloroform method really seems to give me nice 260/280 ratios, so I am just lost right now.

Has anyone run into these problems before or have an ideas as to what may be going on? Any insights or ideas would be GREATLY appreciated.

Thanks in advance...

[mccullou]

I agree with peromhc. And I will add, even when optimized it seems the reaction is VERY sensitive to any changes. One sample will display a good smear another seemingly no digestion. We stayed with sonication

[jasmineja]

I used the enzyme for just testing the fragmentation and was pretty impressed with the fragment size on the gel. It was a focussed smear at 100-600 bp. But when I ran the samples on the bioanalyzer after 2 days, I see a large smear ranging from 30 bp to 2000 bp peaking around 1.5 kb which does not correlate with my gel results!! I'm trying to figure this out by testing some more samples and will update this.

We are using the enzyme on PCR products , the same difference between gel run ( focused smear) and bioanalyzer run, ideas why?. We do purify before run, is it necessary?

[gogreen]

We are using the enzyme on PCR products , the same difference between gel run ( focused smear) and bioanalyzer run, ideas why?. We do purify before run, is it necessary?

I've no clue of why this happens. I tried the enzyme again. This time with a longer enzyme inactivation step. I didnt clean up my reaction. But I still have difference between the gel and bioanalyzer !! I would rather stick to the Covaris or other sonication methods than worry more about this enzyme!! But if it had worked well, life would have been easier!

[aarodriguezm]

Hi you all!!!
Im not working with gDNA but with cDNA and the results are very similar to yours. there is a high variation of fragments although the same sample and diferent incubation times.
I think there is one explanation for the diference between Gel and bioanalyzer.
This product is a combination of two enzymes. one makes some random nicks in both of the DNA strands and the other one recognizes the nicks that are very close to each other and cuts there but leaves another nicks that are not to close to be cut. These uncut nicks reorganize the DNA structure and therefore DNA fragments that are really longer migrate faster than it supposed to do normaly on a gel. The bioanalyzer works like a denaturing gel and measure the real size of your fragments.
You have to experiment with this fragmentase for every single sample. But you dont lose anything because you can do a refragmentation of your already fragmented DNA you only have to clean it up with a column.
If someone have another experience with cDNA fragmentation (not RNA fragmentation) please let me know.
Best regards
Alejandro.

[k-gun12]

Remember that EDTA inhibits the enzyme. Don't elute your DNA in TE or Qiagen AE if you're going to use fragmentase.

[vtosha]

Fragmentase is very comfortable but very capricious enzyme.
We use a half of NEB-protocol's dose of fragmentase and before fragmentation of whole DNA we perform a series of samples with minimum quantity of DNA with different times of fragmentation. Different samples have different reactions to fragmentation but the same sample have the same reaction.
If we use a Qiagen Repli-g Kit or another methods of whole-genome replication, we have a problem: a difficult-for-fragmentation large-size band and band of smaller size. Lower band is fragmentated quickly and higher band - very slowly. So for this samples we use a mechanical framentation.

[rebjk]

Fragmentase is very comfortable but very capricious enzyme.
We use a half of NEB-protocol's dose of fragmentase and before fragmentation of whole DNA we perform a series of samples with minimum quantity of DNA with different times of fragmentation. Different samples have different reactions to fragmentation but the same sample have the same reaction.
If we use a Qiagen Repli-g Kit or another methods of whole-genome replication, we have a problem: a difficult-for-fragmentation large-size band and band of smaller size. Lower band is fragmentated quickly and higher band - very slowly. So for this samples we use a mechanical framentation.

Hi,
I am considering using Repli-g amplification for gDNA prior to whole-genome shotgun sequencing. Is this method working OK for you? Are any modifications needed to proceed with library prep?
Thank you
Barb

[pmiguel]

Also, the phenol chloroform method really seems to give me nice 260/280 ratios, so I am just lost right now.

Just a side note. 260/280 is not a good way to detect phenol. Here is why:



No DNA or RNA there. Just 1 ul of phenol dissolved in 1 ml of ddH2O. The 260/280 is a bit high. But nothing that screams "phenol". Instead you need to look at 270 nm -- that is absorbance maximum for phenol.

The other thing to note is how opaque to UV phenol is. Even at a very low concentration it might be mistaken for a large amount of DNA or RNA.
--
Phillip

[vtosha]

Hi,
I am considering using Repli-g amplification for gDNA prior to whole-genome shotgun sequencing. Is this method working OK for you? Are any modifications needed to proceed with library prep?
Thank you
Barb

We decided to no use Repli-g amplification for whole-genome sequencing. DNA after this is difficult for treatment, no one knows what regions can be lost in spend of Repli-g amplification and fragmentation and amplification in library preparation. We have done viruses' genomes with it, and our results was not very reliable.