Help with Lysis - (Nov/25/2013 )

Of course it's water but I tought I should ask since I am running out of options here. If you think the lysis buffer as it is now should work, than there must be a problem with one of the reageants. Though they all look fine to me.

I could really use some help here. It is frustrating to fail at step 1.

I would try 1% SDS, on the basis that those are the conditions recommended by the suppliers.

Allright, I'll try that Thursday since my access to lab is limited. Than I'll let you know. Thank you very much.

Ahh I must have missed phage434's comment. Sorry about that. Liquid nitrogen is out of question at the moment. But I could try chopping the tip of the feather to small pieces prior to lysing.

By the way here is the paper if it'll be any help: http://vdi.sagepub.com/content/13/2/162.full.pdf

Grinding with dry ice would also work - if you have access to some.

Today I have prepared 4 samples, a new lysis buffer with 1% SDS and new proteinase K aliquouts. This time I chopped feather tips to pieces with sterile razor blades. I preaperd 2 samples each with the original and 1% SDS buffers. And used 5 µg/ml of proteinase K for one of the tubes and 20 µg/ml for the other. So it is:

Lysis Buffer 1 (2% SDS): First tube 5 µg/ml ProK, Second tube 20 µg/ml ProK

Lysis Buffer 2 (1% SDS): First tube 5 µg/ml ProK, Second tube 20 µg/ml ProK

All the samples have been incubating in dry block thermostat at 56C degrees for 4 hours. The samples are as if I just put them there. There is no change. Somehow it feels like proteinase K is not activated at all. I don't know what to do. I'll leave them incubating till tomorrow but I don't expect any changes.

I continue to think that the main issue is the physical size of your fragments. Increasing the surface area accessible to your lysis buffer should be step one.

I will give it a try next week using liquid nitrogen. Though I doubt it's the problem. Because I've never heard of a preparation step like this in any literature about DNA isolation from feathers.

Unlike the paper I shared here, I first soak the samples with 96% alcohol and let it air dry to clean it a bit. Could this have a negative effect in any way?

I just tried a different proteinase K from a completely different source but still no luck. I think I will keep incubating two of the samples over the weekend to see if there'll be any change. Sigma says their proK is fully active in 0,5% SDS. Should I try? Currently I have access to Tris-HCL, EDTA, SDS, Triton X-100. Do you have any recommandation regarding buffer solutions? One that'll work with stubborn tissues and probably quite low DNA yield?

Wouldn't overnight incubation with proteinase K chew up everything? Do you see any debris after the incubation? Try increasing the final conc. of proteinase K a little higher and see what happens.

I used to use a final conc. of 0.6ug/ul of proteinase K and do an overnight incubation at 55C. Every time I would have no tissue (mouse tail) left. I made my proteinase K (from sigma) aliquots with only water and the lysis buffer was made with 0.6% SDS.