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PCR using DNA eluted from agarose gel as a template

PCR using DNA eluted from agarose gel as a template - PCR - Polymerase Chain Reaction Forum

PCR using DNA eluted from agarose gel as a template - 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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  #1  
Old 05-10-2010, 09:34 AM
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Unhappy PCR using DNA eluted from agarose gel as a template



Hi all,
I have a problem with my gel eluted 4kb DNA fragment PCR amplification. After Gel elution I got around 40ng/microliter of DNA concentration and I use 2 microliter out of this for PCR. But I always get a bad smear on my gel picture instead of a specific band. Can anyone help me on how to fix this problem?
My PCR programme is as the following,
1. 94C - 2 min
2. 94C - 45 sec
3. 58C - 45 sec
4. 72C - 4 min
5. Goto step 2 and repeat 29 times
6. 72C - 7 min
7. 4C END
Thanks in advance,
Bests,
DG
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Old 05-10-2010, 11:40 AM
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Default Re: PCR using DNA eluted from agarose gel as a template

Well, at the top of my head I can pull a lot of troubleshooting regarding DNA gel extraction, DNA tempate concentrations, primers and PCR cycle conditions.
For starters I would recommend you to use lower DNA template concentration. You use 80ng per PCR reaction, lower it as much as possible and re-run you DNA extracted in another gel (just a small quantity), in order to check the intensity and putiry of the band. Once run and your band is crystal clear, use your extracted DNA in a lower concentration.
Btw, what is the company of your DNA extraction kit?
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Old 05-14-2010, 12:18 PM
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Default Re: PCR using DNA eluted from agarose gel as a template

I used Qiagen dna extraction kit.
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Old 05-15-2010, 03:46 PM
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Default Re: PCR using DNA eluted from agarose gel as a template

I use a QiaGen gel extraction kit and it works fine for me too. As Drekketh suggested above, the main culprit is likely a high concentration of template.

What I generally do is a dilution starting from 20x. If smears still appear, I increased it to 50x and finally to 100x. Usually by 100x the smears would've disappeared.
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Old 05-16-2010, 10:24 PM
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Default Re: PCR using DNA eluted from agarose gel as a template

I concur, mix up some dilutions and run them and see if it makes a difference.
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Old 06-01-2010, 04:09 PM
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Default Re: PCR using DNA eluted from agarose gel as a template

Agreed... it's likely to be the DNA concentration. Serial-dilute your DNA and re-run your PCR with the dilutions. The more diluted the DNA, the cooler the band you get =D!
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Old 06-11-2010, 08:22 AM
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Default Re: PCR using DNA eluted from agarose gel as a template

Will a high concentration of template inhibit the whole PCR reaction and thus no band formation?
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Old 06-11-2010, 01:00 PM
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Default Re: PCR using DNA eluted from agarose gel as a template

Quote:
Originally Posted by Alicialsw View Post
Will a high concentration of template inhibit the whole PCR reaction and thus no band formation?

Totally. PCR works better the lower the DNA concentration, 'cause polymerase is regulated by product concentration.
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agarose , dna , eluted , gel , gel elution , pcr , smear , template


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