Hi all, undergraduate student here doing research on cyathostomins. I have a very noobish question, so feel free to rip me apart if you take the time to
respond. Learning isn’t always easy, and there is a wealth of experience here.
Simply put, would the DNA from a single C. elegans be visible on a gel without doing any amplification? Or is it simply too small an amount of DNA.
I ask because I am doing DNA extractions in a lysis solution (50 mM NaCL, 2.5 mM MgCl2, 10 mM Tris pH 8.0, 0.45% Tween-20, 100 μg/mL). I am
placing individual cyathostomins in 0.5 mL of this lysis solution with the following temperatures: -20 ℃ for 20 min, 60 ℃ for 3 hours, then 94 ℃ for
15 min.
I was running 0.5% agarose gels to see if I could visualize any DNA so I do not waste PCR reagents, as my funding is limited. So far I have not been
able to see any DNA on the gels, yet the ladders are working. I am trying to determine if my lysis solution is faulty, or if there is simply too little DNA
to visualize before I do PCR. Into my wells I am placing 50 microliters of solution from my 0.5 mL lysis DNA extractions, mixed with 10 microliters of
loading buffer. I have not yet tried to precipitate the DNA.
The C. elegans genome is ~100 Mbp = 10^8 bp.
The molecular weight of DNA is 650 g per mol per bp, so, that’s (10^8 bp) x (650 g/mol*bp) / (6.02 x 10^23 copies/mol) ~ 10^-13 g per copy of the C. elegans genome.
Each adult C. elegans has 959 somatic cells, each with two copies of the genome - call it 2000 copies. An adult will have many hundreds of germ cells, most of them haploid, so let’s conservatively call it 2500 copies of the genome per animal.
2500 copies x 10^-13 g per copy = 2.5 x10^-10 g = 250 pg per animal.
I don’t think you’d see 250 pg on an agarose gel even if it were concentrated in a sharp band - it’s orders of magnitude too low. A randomly fragmented genome won’t be a sharp band.
Under your lysis conditions you are probably more likely to see RNA, particularly ribosomal RNA, before you’d see the DNA.
Also, you don’t mention it, but I assume your lysis solution contains proteinase K?
I appreciate your reply. Yes sorry I forgot to mention the lysis solution does include Proteinase K. I’ve been experimenting with concentrations ranging from 100 micrograms/mL to 300 micrograms/mL.
I’m currently doing some precipitations using ethanol and sodium acetate. I will run them on a gel to see if anything comes up as opposed to just running the lysate.
I think I am going to start doing PCR as soon as my primers come in, as I am really starting to believe that I won’t have an adequate amount of DNA to visualize until I amplify it.
My goal is to amplify the Cya-tbb-1 gene, which I believe is homologous to ben-1. I am quantifying the frequency of benzimidazole resistance providing alleles in cyathostomin populations here in British Columbia.
Why are you trying to visualize the DNA before amplification? From what I understand, your experiment is you’re trying to do single worm PCR using primers probably targeting a particular region of Cya-tbb-1. Once you have the PCR product, how will you do the quantification of resistance alleles? Are you going to sequence the PCR products? Sequencing the products is going to be the most expensive part of your experiment so I wouldn’t be so concerned about using your PCR reagents.
Once you get your PCR reagents and primers (did you design these?), the first thing I would do would be to figure out the optimal cycling conditions to ensure you’re only getting the band of interest.