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PCR product can not migrate into gel

PCR product can not migrate into gel - Agarose Gel Electrophoresis Forum

PCR product can not migrate into gel - Agarose Gel Electrophoresis Forum. A forum to discuss electrophoresis staining, troubleshooting, and development of films for DNA and RNA gels.


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  #1  
Old 08-07-2011, 06:58 AM
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Default PCR product can not migrate into gel



I have tried running the same PCR products on many wells on a 1.0% agarose gel. The products did not migrate into gel, but marker did normally. All the reagents changed, the PCR assay and gel electrophoresis were performed three times more, but the same results got. I am so confused. (gel photo attached)
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  #2  
Old 08-11-2011, 03:33 PM
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Default Re: PCR product can not migrate into gel

Hi and welcome!



Looks like 2 possibilities:
a gel problem or a sample problem

The gel problem could be several issues - recheck your buffer for running, better yet make a fresh one. Also your gel could by accident have been high % but the ladder shows this is probably not the case.

The sample issue could be your DNA is genomic DNA and to run this from experience you need basically way less than 1% ie 0.4-0.7% agarose gel. Your sample could also have impurities that are affecting its running potential.

Any ideas with the above? Do you have extra sample?
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Old 08-13-2011, 01:27 PM
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Default Re: PCR product can not migrate into gel

decrease the concentration of gel
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Old 08-13-2011, 10:48 PM
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Default Re: PCR product can not migrate into gel

Look like Ur PCR reaction it self is not worked. And i donot think there is any problem with your buffer, because ur marker running normally. If you believe your pcr reaction worked then dilute your sample and then run it. One more possibility High salt concentration.

One question, what was the expected product size

Last edited by obama; 08-13-2011 at 10:50 PM.
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Old 08-14-2011, 02:30 PM
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Default Re: PCR product can not migrate into gel

Administrator admin,

Thanks for your cases. The templates used in this PCR assay were purified plasmids, 0.5ul used in per 25ul PCR reaction. I used to use 0.8% agarose gel, unfortunately, it fell to run.
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Old 08-14-2011, 02:36 PM
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Default Re: PCR product can not migrate into gel

to obama,

Thanks. The PCR products size is about 1600bp. How dose the "High salt concentration" contribute to PCR products running on agarose gel electrophoresis?
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Old 08-14-2011, 09:58 PM
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Default Re: PCR product can not migrate into gel

Hi Burnell,

First your answer to admin is not completed, you have to say how much concentration of DNA are you used, if you say 0.5ul (it donot make sense) and how you prepared the DNA either by kit or by Home made buffers.
If you prepared DNA by kit then it is ok but DNA prepared by home made buffers always have high salt concentration (if you not washed it properly). High salt also affect your PCR reaction and also show migration of the PCR products.

And you can cross this link which answer question for me
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if not able see link then copy paste this
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  #8  
Old 08-15-2011, 07:09 AM
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Default Re: PCR product can not migrate into gel

dear
i dont know where to start a new post coz i am new user of this forum so i want to ask that can anybody tell me that which solvent should i use to prepare antibiotics (cofixime and roxithromycin)
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Old 08-16-2011, 03:22 PM
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Default Re: PCR product can not migrate into gel

Quote:
Originally Posted by SAFIA View Post
dear
i dont know where to start a new post coz i am new user of this forum so i want to ask that can anybody tell me that which solvent should i use to prepare antibiotics (cofixime and roxithromycin)
to admin,

thanks for warm advice. The templates used in this PCR assay were purified plasmids by kits. 0.5ul of the plasmids of which the concentration is about < 0.5ng/ul used in per 25ul PCR reaction.
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Old 08-16-2011, 03:26 PM
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Default Re: PCR product can not migrate into gel

Quote:
Originally Posted by SAFIA View Post
dear
i dont know where to start a new post coz i am new user of this forum so i want to ask that can anybody tell me that which solvent should i use to prepare antibiotics (cofixime and roxithromycin)
I am so soory, dear. I am a new one too. You may crosstalk with the any administrators of the forum. Good luck! Sincerely.
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