Always a Band in negative after RT-PCR - RT-PCR and Gapdh (Jun/09/2009 )
I have been carrying out the same experiments for about a month but to no avail,im hoping someone here can help me. I work on Pineal Glands,and i extract RNA using Trizol. One gland generally yields around 90ng/ul.
Usually,after extraction i do DNase treatment and then i do RT-PCR using Superscript III especially designed for qPCR (which is my objective). I extract 2 different genotypes,one 10 day old pineal transgenic with Cdk-1,the other 2m transgenic with Cdk-1. Both follow the same extraction,Dnase,Rt-PCR and even Gapdh PCR.
In my RT-PCR i include a positive and a negative. However,only the 2m negative keeps showing a band after i do a gapdh pcr run. How is that possible? since Genomic DNA was removed by DNase I and the 10d negative was clear? Also,i include an H20-only tube with my gapdh pcr to make sure the contamination was not picked up during that experiment,and it is always giving me a no contamination result with no band.
Any suggestions? Im super-careful with contamination,i UV-treat everything,gloves,tubes,racks,labcoats,pipettes,filtered tips,work under laminar flow hood..etc...
alwaysPCR on Jun 9 2009, 11:33 AM said:
Usually,after extraction i do DNase treatment and then i do RT-PCR using Superscript III especially designed for qPCR (which is my objective). I extract 2 different genotypes,one 10 day old pineal transgenic with Cdk-1,the other 2m transgenic with Cdk-1. Both follow the same extraction,Dnase,Rt-PCR and even Gapdh PCR.
In my RT-PCR i include a positive and a negative. However,only the 2m negative keeps showing a band after i do a gapdh pcr run. How is that possible? since Genomic DNA was removed by DNase I and the 10d negative was clear? Also,i include an H20-only tube with my gapdh pcr to make sure the contamination was not picked up during that experiment,and it is always giving me a no contamination result with no band.
Any suggestions? Im super-careful with contamination,i UV-treat everything,gloves,tubes,racks,labcoats,pipettes,filtered tips,work under laminar flow hood..etc...
Is it more prominent now and then or always the same? What size is the band? Also what are you using to visualize the gel?
It did not used to appear before,my negative RT never used to display a band. Attached you will find a picture of the gel: Ladder - Positive Control - 2 month pineal +RT - Skip 3 wells - 2 month -RT - H20 Blank.
What is odd that in another experiment when i extracted,synthesized cDNA and carried a Gapdh PCR on both 10 day old pineal and 2m old pineal,only the 2m old pineal kept giving me problems with the negative RT.