HydroShear - 8 kb PE library

[bia]

Hi everybody,
to prepare the 8 kb PE library the DNA have to be fragmented to 8 kb of size using the hydroshear, I know this is a critical step.
Do you have suggestion or tips to do this?
Does anyone tried an alternative method to obtain the fragments in the right size range?
Thanks,
bia

[nedoluzhko]

So I have same problem, please help to us...

[pmiguel]

So I have same problem, please help to us...

What problem are you having? Is it that you do not have a Hydroshear? Or is it not giving you the size range that you want.

Typically Hydroshear will give DNA fragments of a size range where the low end is 1/2 the size of the high end. So for 8 kb fragments you would hope to see a range from around 6-12 kb.

If you do not have a Hydroshear, you might use an enzymatic shearing method such as dsFragmentase from New England Biolabs instead.

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Phillip

[Marta]

I tested NEB Fragmentase for generation of long DNA fragments: (http://www.genomecenter.ucdavis.edu/...g_protocol.pdf )

Marta

[pmiguel]

I tested NEB Fragmentase for generation of long DNA fragments: (http://www.genomecenter.ucdavis.edu/...g_protocol.pdf )

Marta

Yes, that shows it does work.

What makes hydroshear great is that it does fragment to a (moderately) tight size range. Fragmentase can cleave anywhere on a DNA strand, so you get plenty of fragments down below 1 kb whereas hydroshear only fragments DNA larger than a certain size. (That size being determined by the "Speed Code", the speed at which the solution is being force through the pore).

Anyway, for many experiments, the amount of DNA will not be limiting. And if you have lots of samples the hydroshear does not scale very well. Whereas enzymatic reactions are easily scaled.

I should add, that back in 1998, during the set up of the Purdue Genomics Core, the hydroshear was probably the only instrument I plugged in, hooked up to a computer and had it perform exactly as specified without tinkering with it for days, weeks or months. (Well, it did require one phone call to GeneMachines to ask how to get rid of a particular sort of bubble.)

There are a few problems one can run into with the hydroshear. But it always seemed to have a much more limited "failure space" to blunder into than most instrumentation I've had to deal with. But it could be my attitude is colored by that initial, easy success back in the days when almost nothing was working right.
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Phillip