The conditions we recommend for running a gel are:
We run a 1% agarose gel. Depending on the size of the gel/number of wells, we load 250 ng to 1 ug of mRNA. We dilute mRNA to 0.2 ug/uL, then add equal volume of NorthernMax Gly sample loading dye.
For making a 1% agarose gel, we would use 50 mL of gel and 0.5 grams of agarose. Microwave the agarose until melted and allow to cool for ~10 min. Add 0.5 uL of ethidium bromide. Swirl gently to mix. Pour gel.
We highly recommend that synthetic, modified long RNAs intended for biological applications are PAGE and HPLC purified. PAGE is better at resolving long, synthetic RNAs while HPLC is critical for removing trace impurities leftover from the PAGE purification process.
Transcript length and integrity are confirmed via agarose gel analysis after glyoxal treatment. Quantity and purity are determined through ultraviolet spectroscopy.
Each template is unique and it is very difficult to predict the outcome of a transcription. Elements such as strong hairpins and repetitive sequences can cause transcriptional stops. If this is a concern, we will perform a small scale transcription and work with you to optimize the transcription reaction for your particular template if needed.
For stocked items, we elute in 10 mM Tris-HCl, pH 7.5. For custom syntheses, we elute in RNase-free water, however we are able to accommodate most special requests.
Our standard purification consists of two silica column steps. We also offer HPLC and PAGE purification.