Help with Lysis - (Nov/25/2013 )

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Hello,

I am trying to use a protocol off a paper to isolate DNA. It's standart phenol:chloroform:iaa extraction followed by ethanol precipitation. So far I haven't been successful. I think the problem is in lysis step. Because the paper claims that sometimes samples completely dissolve in lysis buffer. But mine don't. Furthermore, when I proceed to phenol:chloroform:iaa I see no interphase indicating no protein. The lysis buffer in the paper is as follows:

"500 µl of lysis buffer (50 mM Tris-HCl, pH 8, 20 mM ethylenediaminetetraacetic acid , pH 8, 2% sodium dodecyl sulfate) and proteinase K at a final concentration of 175 µg/ml."

I take the necessary amounts of Tris-HCl and EDTA from stock solutions and fill it up to 500 µl with 2% SDS. Than I add 4 µl of 20 µg/ml Proteinase K solution.

What seems to be wrong?

Thanks.

-burakkc-

You didn't say what organism/tissue you are lysing. This makes a big difference.

-phage434-

Well I didn't think it would be important because it's the same material with the paper but you're right I should've. I'm trying to isolate DNA from the calamus of naturally molted feathers.

-burakkc-

And the incubation conditions for the proteinase K step?

-bob1-

Tried 56C for 4 hours, 37C overnight and 56C overnight. None of 'em worked.

*Paper says 56C for 4 hours is good for small samples, while bigger samples should be incubated at 37C overnight.

-burakkc-

I should also note that, I tried double and triple the amount of proteinase K and unfortunately still no result.

-burakkc-

Having had a look at a number of digestion protocols just now - it seems that 1% SDS is the usual concentration. 2% might be too much.

-bob1-

Well the paper claims that the protocol worked for over 600 samples from different birds. There is no ddH2O on the buffer recipe as you can see. So I simply fill it up with %2 SDS to rech desired volume. Is this right? And another point I think I might be doing wrong is the proteinase K concentration? Is my calculation correct?

-burakkc-

Are you pulverizing your sample prior to digestion? A bead beater or grinding after LN2 freezing would probably do the job. I would guess that unless fine particles are used, it will take a long time to lyse this tissue.

-phage434-

It is a well known thing that people often don't put the full (or sometimes incorrect, intentionally or unintentionally) methods in their papers, mostly in the interests of space saving. So I am not surprised that there is something not working.

What do you think the diluent is for your SDS - if it isn't water, I will be very surprised!

-bob1-

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