Hi All
Has anybody ever seen anything like these?
I'm asking this question in as many forums as possible since nobody around me seems to have any clue what is going on - even the techs at Agilent say they've never seen traces like these.
They are total RNA isolations. I have troubleshot this issue several times, thought that I'd solved the problem, only to have it crop back up again and be solved again.
Please assume that i am following the Agilent manual fairly closely at this point and have even added steps suggested by Agilent techs from my previous troubleshooting sessions.
Earlier solutions that seemed to have fixed the problem before include extra time to equilibrate all reagents to room temp, vortexing the prepped chip at 2000 rather than 2400, and allowing extra time for the electrodes to fully dry after RNase-free water wash. The same samples that gave me problems, would then read just fine once each of these changes in protocol were made. I have also tested for vibration from a nearby centrifuge and shearing of the gel by high speed centrifugation.
Several other people, from several other labs are using this same machine [often times in between 2 of my runs] and nobody is getting anything even close to this. The issue is absolutely something I'm doing wrong with my prep.
The attached photos are recent, from one of four runs using the same samples. No sample gave the same results twice in any of the four runs. Clearly the samples are not the problem.
HELP!
Thanks
Has anybody ever seen anything like these?
I'm asking this question in as many forums as possible since nobody around me seems to have any clue what is going on - even the techs at Agilent say they've never seen traces like these.
They are total RNA isolations. I have troubleshot this issue several times, thought that I'd solved the problem, only to have it crop back up again and be solved again.
Please assume that i am following the Agilent manual fairly closely at this point and have even added steps suggested by Agilent techs from my previous troubleshooting sessions.
Earlier solutions that seemed to have fixed the problem before include extra time to equilibrate all reagents to room temp, vortexing the prepped chip at 2000 rather than 2400, and allowing extra time for the electrodes to fully dry after RNase-free water wash. The same samples that gave me problems, would then read just fine once each of these changes in protocol were made. I have also tested for vibration from a nearby centrifuge and shearing of the gel by high speed centrifugation.
Several other people, from several other labs are using this same machine [often times in between 2 of my runs] and nobody is getting anything even close to this. The issue is absolutely something I'm doing wrong with my prep.
The attached photos are recent, from one of four runs using the same samples. No sample gave the same results twice in any of the four runs. Clearly the samples are not the problem.
HELP!
Thanks
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