I'm using 0.7% agarose gel for elution of restriction digested plasmid DNA and my insert ranging from 2.3 kb to 5.7 kb from two different brands having gel strength >1200g/cm2 (1.5% ) and >2200g/cm2 (1.5% ). Inspite of trying all available tips suggested on Rsearchgate and other internet sources I'm continuously getting DNA concentration between the range of 5-10 ng after elution even for digesting 10 ug DNA. I'm just wondering is agarose can be a culprit in my case. Kindly help.
I doubt it's the agarose honestly, especially if you're seeing the expected bands very clearly under illumination. Are you using a kit with silica columns and a buffer to melt the gel or are you using another method to extract DNA?
If you're using a silica column kit, my bet would be on two issues:
1. Salt contamination, the chaotrope commonly used to dissolve the gel is very fluorescent around the same wavelength as DNA. Adding extra washes and letting the desalting buffer sit on the column for a few minutes can help this sometimes. Other times, you can just run the eluted product through a PCR clean-up kit and get clean, measurable DNA. I've had gel extracted DNA that goes from "7 ng/µL" to around 70 ng/µL just by using an additional clean-up step. If you're getting a low 260/230 ratio then it's probably this.
2. The kit is old. If the dissolving buffer has turned obviously orange/yellow then it's probably pretty oxidized and in my experience that leads to a lot of issues when dissolving the gel. Other issues can happen like evaporation of the alcohol in the wash buffers too, but typically the dissolving buffer goes bad before this. Gel extraction kits tend to have a shorter shelf life than PCR clean-up kits do. The only real solution here is to buy a new one.
If you're using another way then I don't have any good suggestions, I've never gotten methods like "freeze & squeeze" extraction to work for me.
Hi, what's your downstream application? In most cases, there is a way to avoid the gel extraction step, which is in my eyes pretty much the worst thing one can do with DNA.
From what I remember, having a very high concentration of agarose means that you should add excess of the first buffer to make sure the gel completely dissolves. Minimizing the size of the gel slice also helps. Non-dissolved agarose means that you'll clog up the column membrane.
I don't think agrose gel is the problem. In my lab, we only use one method to purify everything, inserts, plasmid, etc. We use just melt the agarose piece containing desired insert, digested plasmid, etc in 3 times volume of Binding Buffer (Wang et al., 2011 - http://www.funpecrp.com.br/gmr/year2011/vol10-1/pdf/gmr1055.pdf), transfer to column, centrifuge, wash with 75% ethanol and elute. It works every time. Good luck!
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