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PCR no band

PCR no band - Molecular Biology Techniques

PCR no band - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 03-04-2014, 02:48 AM
Nur Nur is offline
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Exclamation PCR no band



Previously I have carry out an optimization of pcr by varying MgCl2 concentration (1.0 mM, 1.5 mM, 2.0 mM) two tubes for each different concentration. After run agarose gel electrophoresis, all 6 wells show band and some has multiple band. Next, I reamplify pcr product from tube containing 1.0 mM MgCl2. I viewed the gel, unfortunately there is no band. Plus, dilute a primer. Is there any problem with my primer dilution?
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Old 03-04-2014, 04:46 AM
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Default Re: PCR no band

Nur, welcome. Your details on the problem is a bit unclear and I don't want to make assumption here.

Why not just re-run the PCR with 1.0mM MgCl2? This time include your original concentration primer and the diluted primer.

Do let us know how strong the band is as most time too much DNA will KILL PCR.
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Old 03-04-2014, 04:38 PM
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Default Re: PCR no band

Hi Nur, I would agree with Butters response concerning running a parallel PCR using the original concentration of primers alongside the diluted primers. It also depends on the concentration of the PCR that you used to re-amplify the second time. Depending on the original source of template for the first PCR, a small PCR fragment would have far more copies available for amplification then genomic or plasmid DNA. For example, a 500 bp PCR product would need very little DNA for the second amplification.

You may also want to run the original PCR reaction alongside the second set of PCR as a positive control. By using a positive control you would be able to confirm that there is nothing wrong with any of the reagents used to set up PCR.
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