Another 'please help me with my sonication' post - (Feb/22/2007 )

Hi,

In continuation from a previous post of mine:

I am am interested in the acetylation state of histones associated with immune genes that are expressed in brain. My protocol includes;

Prepping tissue sample

Douncing ~100mg tissue in 5ml 1x PBS
Fixing with formaldehyde (1% [final]) for 10 min at RT.
Quench with 2.5M Gly (0.125M gly[final]), then spin and wash pellet.
Lyse the pellet with an SDS lysis buffr supplemented with proteinase inhibitors

I then Sonicate,

Being the first time I've tried this technique I used a variety of sonicating procedures. I use a Sonics Vibracell 130, with a microprobe tip (2mm) set at 30% amplitude. I sonicate on ice.

I've tried sonicating 8-20x 10 sec in length with a 60sec rest period between probes.

After sonicating I reverse crosslinks (4h @65 with 5M NaCl), remove protein and RNA, then isolate DNA with phenol-chloroform. I managed to isolate a very small amount of DNA (20-50ng/ul)

So I put my weak isolate on a 2% gel and what I see is even more disconcerting: see attached image

Here are my questions:

Why is my DNA concentration so small, where am I losing DNA ?

What are these smears? (The white bands at the bottom are loading buffer, the ladder is 100bp)

Have I sucessfully, if minimally , sheared any DNA?

-bosco-

QUOTE (bosco @ Feb 22 2007, 03:11 PM)

?

What are these smears? (The white bands at the bottom are loading buffer, the ladder is 100bp)

Have I sucessfully, if minimally

, sheared any DNA?

Have you tried increasing the amplitude of your sonication. There is the heating problem but, in my case, I've gotten good results even when the tube is warm to the touch after sonication (and I sonicate on an ice bath).

As for the DNA yield you're getting, what is the total amount of DNA yield per 100mg tissue. Do you know that you are getting a lower amount of DNA than you would expect by another extraction method. In the case of the brain, the area you are using might have a lot of axons and thus might not have as many nuclei per mg of tissue as, for instance, the liver.

-KPDE-

Hi, and thanks for the quick response

I would normally expect 850-1600 ng/ul of DNA from 100mg brain tissue, assuming DNA is dissolved in 100 ul ddH2O. I'm at about 10% efficiency.

As this was a test prep I used the entire brain, 100mg per sonicate rxn..

I've been thinking I should lower the concentration of formaldehyde to 0.8%, and maybe reducing the fixing time. I could adjust the amplitude of the sonicator too.

-bosco-

Hi,

Quick update:

So I noticed out that the sonicator I was using (Sonics-135vx) has an output of 135W, not very high. At 30% amplitude I figure the output was ~35W. I also noticed that other posters on this board use sonicators with numbers like brandx650, or brandY450. The 650 and 450 are wattage? So I used a new machine a vitacell(or something similar) 600, 600Watts!! Yeah, thats power!! So at 25% amplitude I sonicated my samples between 10-15x with 10 sec bursts. I also fixed with two different % ( 0.8 or 1.0%) of formaldehyde, and for different lengths (5, 10, or 15 min).

I still have a very low concentration of DNA after PIC extraction, (2ug from 50mg brain tissue)...

Gel images are attached, I hope this info helps....

-bosco-

Hi bosco

Looks like you found the problem with the sonicator - in the first picture (from Feb. 23) there is no sheared DNA visible.
after (successful) sonication you get a smear of (much smaller) bands between 100 and 600 bp - looks correct!

The smears on the gel might be salts - checked for that?

Your DNA yield is very low though, should be more like 50 µg form 50 mg tissue, from brain a bit less, maybe 25 g from 50 mg tissue.
Could you maybe post a more detailed description of your isolation process?

-PFA-Goofy-