I am working on MRE-library construction, which basically requires cutting the gel slices of 220-600bp lengths. I attached my gel picture here, it was after PCR amplification and before gel cutting. My six samples were located on the 3rd, 5th, 7th, 9th, 11th, and 13rd lanes, respectively. It looked that all my 6 samples were ok, I had got plenty of DNA fragments amplified of 200-600bp lengths.
I cut the gel and extracted DNA using MinElute columns (1 column for each sample), and eluted the DNA in 16ul EB buffer, then I quantified the DNA concentration by Qubit Quant, and it showed that concentrations were all very low. For sample1 through sample4, the concentrations were all around 20-30ng/ML, and for sample5 and sample6, the concentrations were a litter better, both were of about 100ng/ML, but all the concentrations were much lower than our expectation (usually, the concentration should be 200-300ng/ML).
I also checked the DNA samples in the nanodrop machine, and I checked the peaks, and it showed that there were no peaks at 260 at all, does that mean my DNA samples were contaminated?
I thought, by looking at the gel picture that I attached here, we should be able to get plenty of DNA extracted. Can anyone tell me your thoughts on how this could happen.
I am pretty new at bench work, I have done a lot of computational work, but not much on experiments, so I do have some difficulties figuring out the reasons for problems like these.
Well gel extraction is always difficult, one thing I found is that switching to other stain than ethidium bromide (I use sybrgreen) and a blue lamp instead of UV, combined with minelute was really working well.
Hi Jackie,
I think your DNA concentration after gel extraction is okay. Sometime I get even lower concentration and it works fine afterwards......the most important thing is what is the next step u want to follow.............I will suggest to use higher PCR reaction as Kris said and run on a gel.......for 50 ul sample use normal 10 wells or u can combine two wells...don't use much wider wells as it will increase the gel amount. Also try to keep the column after adding EB buffer or heated water for 1-2 mins before centrifuging...this increase yield......Best of Luck :)
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