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Help with Lysis


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#1 burakkc

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Posted 25 November 2013 - 04:52 AM

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 [EDTA], 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.

 

Edited by burakkc, 25 November 2013 - 04:53 AM.


#2 phage434

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Posted 25 November 2013 - 09:17 AM

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



#3 burakkc

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Posted 25 November 2013 - 11:53 AM

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.



#4 bob1

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Posted 25 November 2013 - 11:56 AM

And the incubation conditions for the proteinase K step?



#5 burakkc

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Posted 25 November 2013 - 11:59 AM

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.


Edited by burakkc, 25 November 2013 - 12:05 PM.


#6 burakkc

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Posted 25 November 2013 - 12:01 PM

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



#7 bob1

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Posted 25 November 2013 - 12:18 PM

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.



#8 burakkc

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Posted 25 November 2013 - 12:25 PM

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?



#9 phage434

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Posted 25 November 2013 - 12:44 PM

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.



#10 bob1

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Posted 25 November 2013 - 12:53 PM

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!



#11 burakkc

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Posted 25 November 2013 - 12:58 PM

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.



#12 bob1

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Posted 25 November 2013 - 01:07 PM

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



#13 burakkc

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Posted 25 November 2013 - 01:39 PM

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.


Edited by burakkc, 25 November 2013 - 01:50 PM.


#14 burakkc

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Posted 25 November 2013 - 01:55 PM

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



#15 bob1

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Posted 25 November 2013 - 02:19 PM

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






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