Hi
I am trying to amplify linearized circular ssDNA molecules by PCR using primers. when i run the PCR products on an agarose gel, I am able to see the right molecular weight band (~120bp) but also high molecular weight bands on the agarose gel near the loading wells (please see the lanes to left of the ladder on the gel). I dont see these products when i amplify ssDNA that is only linear (please see the lanes to the right side of the ladder).
Any comments regarding these high molecular weight bands near the loading wells would be much appreciated.
Thanks
This ban that you see in the gel can be part of your template DNA that is not amplifying (if you see, your bands at 120 bp on your left are smother than on your right). How much DNA did you load on the gel? Another possibility can be that polymerase is stuck to your amplicon. If you load the DNA with SDS could be solved the problem.
Here another person had the same problem with his PCR: https://www.researchgate.net/post/PCR_product_retains_in_agarose_gel_well_during_electrophoresis-any_thoughts
Probably can be helpful for you.
Depending on your circularization procedure, it could be intermolecular linkages, rather than intramolecular linkages. However, circularized ss DNA should have a different gel mobility than linear ds or ss DNA. I would be hesitant that the 120 bp material on the left is your circularized ssDNA since it runs identically as the non circularized material on the right.
This ban that you see in the gel can be part of your template DNA that is not amplifying (if you see, your bands at 120 bp on your left are smother than on your right). How much DNA did you load on the gel? Another possibility can be that polymerase is stuck to your amplicon. If you load the DNA with SDS could be solved the problem.
Here another person had the same problem with his PCR: https://www.researchgate.net/post/PCR_product_retains_in_agarose_gel_well_during_electrophoresis-any_thoughts
Probably can be helpful for you.
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