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Topic: Question about PCR yielding blurry sites under electorphoresis  (Read 10231 times)

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Offline minimal

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I was given a question today that said what would you do, if you performed a PCR reaction and you didn't notice clear bands under electrophoresis.  I answered that possibly the primer was not binding to the correct site, that it was doing some wobble bonding.  After that, I was asked whether or not whether or not I would raise or lower the temperature in order to provide optimal conditions.  I said I would lower it, which was apparently incorrect.  My reasoning though is that the wobble bonds would be harder to maintain under a lower energy environment, but obviously this is not correct, could someone explain this a bit more to me.
In addition, apparently another way to answer the question is that the introns are being produced on the electrophoresis, and you can use an enzyme to remove them (forget the name of it off hand). Could someone also explain why the occurrence of introns would produce such a blurry band?
Thanks

Offline Yggdrasil

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Re: Question about PCR yielding blurry sites under electorphoresis
« Reply #1 on: July 31, 2007, 05:46:50 PM »
Temperature is used to control the stringency of primer binding.  The stronger the primer-template bond, the higher temperature it can withstand.  Therefore, increasing the temperature will cause weak primer-template associations to break apart.  Since wobble binding is weaker than watson-crick binding, these wobble bound pairs would break off at higher temperatures while the watson-crick binding would remain stable.

I'm not sure about why introns would produce blurry bands.

Offline sdekivit

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Re: Question about PCR yielding blurry sites under electorphoresis
« Reply #2 on: July 31, 2007, 06:00:00 PM »
introns contain many repetitive sequences so when u use short primers, the chance is very likely that the primer binds an intron sequence at different places, causing blurry bands.

Offline minimal

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Re: Question about PCR yielding blurry sites under electorphoresis
« Reply #3 on: August 06, 2007, 06:04:04 PM »
great guys thanks for the info

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