Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Agarose Gel Electrophoresis Forum (http://www.molecularstation.com/forum/agarose-gel-electrophoresis-forum/)
-   -   PCR product can not migrate into gel (http://www.molecularstation.com/forum/agarose-gel-electrophoresis-forum/83311-pcr-product-can-not-migrate-into-gel.html)

Burnell 08-07-2011 06:58 AM

PCR product can not migrate into gel
 
1 Attachment(s)
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)

admin 08-11-2011 03:33 PM

Re: PCR product can not migrate into gel
 
Hi and welcome!

http://www.molecularstation.com/foru...-gel-snap1-jpg

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?

SAFIA 08-13-2011 01:27 PM

Re: PCR product can not migrate into gel
 
decrease the concentration of gel

obama 08-13-2011 10:48 PM

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

Burnell 08-14-2011 02:30 PM

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.

Burnell 08-14-2011 02:36 PM

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?

obama 08-14-2011 09:58 PM

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
[Only registered and activated users can see links. Click Here To Register...]

if not able see link then copy paste this
[Only registered and activated users can see links. Click Here To Register...]

SAFIA 08-15-2011 07:09 AM

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)

Burnell 08-16-2011 03:22 PM

Re: PCR product can not migrate into gel
 
Quote:

Originally Posted by SAFIA (Post 435691)
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.

Burnell 08-16-2011 03:26 PM

Re: PCR product can not migrate into gel
 
Quote:

Originally Posted by SAFIA (Post 435691)
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.


All times are GMT. The time now is 03:49 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.09830 seconds with 11 queries