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#1
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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 __________________________________ Do you Yahoo!? Yahoo! Small Business - Try our new resources site! http://smallbusiness.yahoo.com/resources/ --- |
#3
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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 --- |
#4
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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 --- |
#5
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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 Quote:
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#6
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Correct web address is www.cartagen.com
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