I'm trying to establish if a mutation performed on a specific gene worked. I know the size of my gene in question and will eventually send it for sequencing. I don't need to clone it just sequencing.
I was instructed by my PI to run an agarose gel with no EtBr, in other words to not use UV light to visualize the DNA position as to avoid sticky ends formation and somehow cut the section where the gene is supposed to be and the stain. I guess there are ways to go about this but am wondering if a protocol already exists that I can be referred to?
I presume this is a PCR product
1. in case you have a clear single band amplification then the PCR product can be sequenced after treatment with exonuclease and shrip alkaline phosphatase, most commercial sequencing facilities offer PCR product purification for a small charge, gel elution is required only when there are non-specific amplification
2. There are safe dyes such as methelene blue that stain the DNA based on charge and can be visualized as a blue coloured band in a light blue background, some commercial formulations are also available, but i dont know how this will affect sequencing.
Hi Alex,
Very good suggestions from Vijayakumar.
I am not sure why you want to avoid staining (EtBr) or using any alternative safe staining. There are a few stains such as GelGreen Nucleic Acid Gel Stain that doesn't need UV light to visualize.
But in any case if you have to do it your way, I would guess you can run some of your sample into a different well on the same gel (not far away from the well with your main sample), then cut the gel into two halves, and stain one half by immersing your gel in a normal running buffer with some EtBr for 20 min or so.
Visualize where your band on the stained half, then put them next to each other and try to cut a position on the same line with your stained band. This of course assuming your gel was running nicely and in straight line.
Good luck
Hi Alex,
I liked the suggestions before, but think that is much work to do this. If you do not have dye which does not interact with DNA (like gentian violet, wich is the best one), you should try to separate using another method. Separate your DNA using another method, like Size Exclusion chromatography, if your insert is larger enough or still try capillary electrophoresis, using a timer and testing eluted fractions by time.
If you wanna try another techniques, you can use:
- this reagent (http://www.amresco-inc.com/Visual-Violet-DNA-Gel-Kit.cmsx)
or use techniques of both attached articles.
Best wishes.
I don't understand what you are trying to do! Please provide more explanation.
As someone already pointed out above, your question is unclear. If you want to see whether the PCR worked or not, you simply run a small fraction on an agarose gel and purify the rest of PCR product by using Qiagen PCR product purification kit or similar kind from other companies before sending for sequencing. If you are trying to create mutations in a plasmid, run a sample on an agarose gel to confirm whether PCR worked or not and then transform it into E. coli, isolate plasmid from single colonies and send them for sequencing.
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