I have qubit confirmation that my pcr produced 21 ng/ul of dsDNa in a 50ul reaction volume. I chose primers for a specific gene/sequence of 1.4kb . But my gel electrophoriesis shows a nice ladder, but no bands from my sample DNA, of which I used 2 ul with loading dye. I put etbr in the gel and the buffer. I have made 5 attempts now with varying concentration of agarose, up to 2%. I only ever see the ladder under UV. I don't think it possible the qubit is wrong, the dye it uses bonds only to dsDNA and ignores any contaminates. Do I have no DNA or am I just doing this gel incorrectly? My dye mostly vanishes within minutes of starting the gel run. thank you ! - Amy J.
tell me about your loading dye makeup please?
It is possible if you do not add sucrose or glycerol to weigh down the dna sample then it will all float out of the well and nothing will be visible. How much dna are you loading ?
loading 40 ng of DNA in 2ul of volume, loading dye is supposed to have glycerin in it. vortexed dye before using it.
Hi Amy
About the vanishing of your dye, did it happen to your ladder as well? Have you used the same loading for you ladder and PCR products?
I can see the ladder. Going to try a run now with 1 ul glycerol added and maybe .5 ul methylene blue.
add more glycerol ( you cannot have too much glycerol) or borrow loading dye from another researcher or even add your dna to the ladder and look for an additional band
this is strange - it looks like my dye ran the wrong direction! it flowed into the black fill tank, which has bubbles. It is not running to red, the black fill tank is all blue and the wells have almost disappeared. odd....
OK. it looks like the cheap Chinese power supply I bought is doing.... bad things. I flipped the electrodes, and I got nice bands... running red to black.
bizarre I would have expected the ladder to run backwards as well in this situation not just the dna samples. Thank you for letting us all know the solution
I was able to run a new ladder and I got 1 band as expected in the right place from my DNA sample. Buying a new PS today!
Can the Qubit be wrong? Can I get 30 ng/ul qubit readings after PCR, and still fail to get a band on the gel, when the ladders develop perfectly? I loaded the well with 5 ul of 30ng/ul DNA samples and got only ladders. again.
update : I ran a known 3kb control and what I think is my 3kb cassette i have been laboriously constructing using 5 different restriction enzymes! but they rode much higher than the 3kb spot on the ladder, left them running about an hour.
they are level with each other.
the only picture attached here is one taken from the underside of the gel after i cut out most of the band from the front side.
the ladders are much more visible from the back side of the gel.
not the best gel, but I am improving! and single bands tends to indicate my 6 pieces are all in the cassette, no primer dimer or junk
observed, just 2 clean bands.
i used less loading dye than the protocol i had called for, and I got bands! made no other changes. 1 less ul.
attached iphone heic image file
the front side initial pictures all came out blurry and bad.
here is a photo of this gel
curious as to why the bands outran the ladder!?
huh maybe i just measured small fragments in those two bands? they are not close to the wells ;-(
I went back to the original pcr's I did (4) from the twist synthetic dna and I got nice results for these.
these are the various pieces of the cassette before adding RE with PCR, and I think I know how i got primer dimers in those.
going to start that over, get it right.
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