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Mike Kowalski

Mike Kowalski - PCR - Polymerase Chain Reaction Forum

Mike Kowalski - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 04-28-2007, 02:33 AM
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Default Mike Kowalski



I'm currently looking at some microsatellites in a species of sunflower. Initially, we extracted DNA from seeds, which has worked nicely. Getting suitable DNA from leaf tissue has been more difficult. I'm guessing that there's secondary compounds in the leaf tissue that are interferring with the pcr reaction.
We recently ran some samples of DNA derived from leaves using different extaction protocols. Some of these were successful in getting microsat amplification. However, we observed something odd, and I was wondering if anyone might have a possible explanation.
In the reactions that resulted in amplified microsats we also got primer dimers. In reactions that did not result in amplified microsats we got no primer dimers. I'm puzzled. What could be going on here?
Thanks.
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Old 07-23-2007, 03:45 PM
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Default Re: Mike Kowalski

Quote:
Originally Posted by m.kowalski View Post
I'm currently looking at some microsatellites in a species of sunflower. Initially, we extracted DNA from seeds, which has worked nicely. Getting suitable DNA from leaf tissue has been more difficult. I'm guessing that there's secondary compounds in the leaf tissue that are interferring with the pcr reaction. .
Secondary compounds like starches, phenolic compounds. Old school method is to have a salt precipitation step using 5M KCl--worked great on arabidopsis, new method is use a kit like Qiagen DNA Plant Extaction kit.

Quote:
Originally Posted by m.kowalski View Post
We recently ran some samples of DNA derived from leaves using different extaction protocols. Some of these were successful in getting microsat amplification. However, we observed something odd, and I was wondering if anyone might have a possible explanation.
In the reactions that resulted in amplified microsats we also got primer dimers. In reactions that did not result in amplified microsats we got no primer dimers. I'm puzzled. What could be going on here?
Thanks.
Repeat the procedure, if the conundrum reoccurs, then analyze and optimize beginning with primer concentration, number of cycles. Don't drive yourself crazy unless the problem repeats everytime you PCR.
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