Hello,
I am trying to do siRNA complexation with simple PEI of 25kda first and my synthetic co-polymer afterwards. I am facing problem in initial phase. When I checked nano-drop values of my scrambles (i.e. Negative control siRNA) it is giving negative values and negative concentration . Also A260/A280 ration is also around 1.414 with pure siRNA which gradually becomes negative when I added my different polymer ratio to it. Even I have tried gel retardation assay to check complexation by using 1%w/v agarose and still I am nit getting any single bands which means either my RNA is not getting stain or my gel pore size is very big for 21bp siRNA.
Please suggest me anything to counteract the same.
All spectrometers but especially nanodrop are entirely dependent on having a perfet blank ( zero) sample to compare samples against. So, for instance, you cannot zero on water and meaure in TE. They must have the same absorbance and the same pH and measurement must be quite quick or absorption of CO2 from the air will change the PH and then the readings. I would clean the optics of the nano with acid as recommended in the manual in case the optics are coated with protein or dna from previous samples. Also zero each stage of the reaction appropriately so if you are adding a reagent in another solvent then the same reagent should be added to the new zero or the zero and sample are not directly comparable
Dear Paul sir,
yes I have used same diluent to zero and then I have added my samples. Like nuclease free water as blank for my sirna and HEPES buffer pH.7.4 for my complexes.
I would avoid using nanodrop. The Qubit RNA kits are much more sensitive and reliable in quantifying short oligos, specially in the presence of contaminants such as your polymers.
Thank you Hasan Issa. I am also trying to use ELISA plate reader for checking absorbance of my samples.
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