DNA isolation from old leaves
 

Hi everybody,
I am having trouble isolating DNA from oak leaves that
went from the tree into the -80C freezer about one
year ago. Nothing else has been done with them. I am
using the Qiagen Plant DNA kit so far, but PCR works
only occasionally. Does somebody have a DNA isolation
protocol which might give a better chance of getting
PCR products?
thanks a lot for your help,

Manuel



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---

(some body) wrote in
o.com:

Hi everybody,
I am having trouble isolating DNA from oak leaves that
went from the tree into the -80C freezer about one
year ago. Nothing else has been done with them. I am
using the Qiagen Plant DNA kit so far, but PCR works
only occasionally. Does somebody have a DNA isolation
protocol which might give a better chance of getting
PCR products?
thanks a lot for your help,

Manuel



__________________________________
Do you Yahoo!?
Yahoo! Small Business - Try our new resources site!
http://smallbusiness.yahoo.com/resources/
---


Hi Manuel,

Qiagen states in their handbook that oak leaves result in a very viscous
lysate with huge amounts of precipitates which may give sheared DNA if
directly loaded onto the shredder columns. They recommend to introduce an
additional centrifugation step (handbook page 13). Did you try that?
So far, we didn't have much luck using commercial kits for grapevine
leaves (rich in polysaccharides and phenolic compounds - same as oak?).
Just right now I am testing another bunch of products for that purpose.
But so far it was always "back to the home brew" ...

Cheers,

Klaus

Hi! All,

Try the conventional CTAB method with higher concentration of NaCl. 1.5M
NaCl is suggested for the first time.

Wen
----- Original Message -----
From: "Dr. Klaus Eimert"
To:
Sent: Monday, April 18, 2005 3:18 AM
Subject: DNA isolation from old leaves


(some body) wrote in
o.com:

Hi everybody,
I am having trouble isolating DNA from oak leaves that
went from the tree into the -80C freezer about one
year ago. Nothing else has been done with them. I am
using the Qiagen Plant DNA kit so far, but PCR works
only occasionally. Does somebody have a DNA isolation
protocol which might give a better chance of getting
PCR products?
thanks a lot for your help,

Manuel



__________________________________
Do you Yahoo!?
Yahoo! Small Business - Try our new resources site!
http://smallbusiness.yahoo.com/resources/
---


Hi Manuel,

Qiagen states in their handbook that oak leaves result in a very viscous
lysate with huge amounts of precipitates which may give sheared DNA if
directly loaded onto the shredder columns. They recommend to introduce an
additional centrifugation step (handbook page 13). Did you try that?
So far, we didn't have much luck using commercial kits for grapevine
leaves (rich in polysaccharides and phenolic compounds - same as oak?).
Just right now I am testing another bunch of products for that purpose.
But so far it was always "back to the home brew" ...

Cheers,

Klaus




---

Hi! All,

Please check out this paper:
http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb16/16069-1.pdf
The first method described seems to be the best one.

Wen
----- Original Message -----
From: "Dr. Klaus Eimert"
To:
Sent: Monday, April 18, 2005 3:18 AM
Subject: DNA isolation from old leaves


(some body) wrote in
o.com:

Hi everybody,
I am having trouble isolating DNA from oak leaves that
went from the tree into the -80C freezer about one
year ago. Nothing else has been done with them. I am
using the Qiagen Plant DNA kit so far, but PCR works
only occasionally. Does somebody have a DNA isolation
protocol which might give a better chance of getting
PCR products?
thanks a lot for your help,

Manuel



__________________________________
Do you Yahoo!?
Yahoo! Small Business - Try our new resources site!
http://smallbusiness.yahoo.com/resources/
---


Hi Manuel,

Qiagen states in their handbook that oak leaves result in a very viscous
lysate with huge amounts of precipitates which may give sheared DNA if
directly loaded onto the shredder columns. They recommend to introduce an
additional centrifugation step (handbook page 13). Did you try that?
So far, we didn't have much luck using commercial kits for grapevine
leaves (rich in polysaccharides and phenolic compounds - same as oak?).
Just right now I am testing another bunch of products for that purpose.
But so far it was always "back to the home brew" ...

Cheers,

Klaus




---

Hi Everyone,

You might want to check out the Cartagen DNA Extraction Kit for Plants. It combines a CTAB method with a column clean up step. They also have some kits for going straight to PCR that have been used on oak leaves. Their product info is at www.cartgen.com.

dwm

Correct web address is www.cartagen.com