Smear On SYBR Gold stained Gels.

marlon4711 · 11-06-2015, 08:04 AM

Hey everyone,
I wanted to try to pick your brains on this this phenomenon that I often observe but can't really wrap my head around:
I often observe a very long smear on SYBR stained PAGE or E-gels after too many PCR cycles of small RNA libraries. (See attached picture 8, 10, 12, 14 cycles.)
It only happens when the library is getting 'overamplified'. The smear seems like some sort of artifact to me because it reaches to several 10s of kb and I use only an extension time of 10sec. I am also unable to TOPO clone 'fragments' in the smear of several 100 bp.
Is this some sort of concatemerization of the PCR products? Or simply a smear from overloading the lanes?
Thanks and all the best,
Marlon

nucacidhunter · 11-06-2015, 05:26 PM

Attachment has not come through.

Are you able to TOPO clone smaller fragments from the same gel cut or libraries?

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11-06-2015, 08:04 AM	#1
marlon4711 Junior Member Location: USA Join Date: Nov 2015 Posts: 2	Smear On SYBR Gold stained Gels. Hey everyone, I wanted to try to pick your brains on this this phenomenon that I often observe but can't really wrap my head around: I often observe a very long smear on SYBR stained PAGE or E-gels after too many PCR cycles of small RNA libraries. (See attached picture 8, 10, 12, 14 cycles.) It only happens when the library is getting 'overamplified'. The smear seems like some sort of artifact to me because it reaches to several 10s of kb and I use only an extension time of 10sec. I am also unable to TOPO clone 'fragments' in the smear of several 100 bp. Is this some sort of concatemerization of the PCR products? Or simply a smear from overloading the lanes? Thanks and all the best, Marlon

11-06-2015, 05:26 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,248	Attachment has not come through. Are you able to TOPO clone smaller fragments from the same gel cut or libraries?