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Smog187, on 03 October 2012 - 11:12 AM, said:
Maybe you are not adding enough Ethidium Bromide? Your samples could have run off the gel? Are your DNA fragments at a high enough concentration to be seen?
My ladder does show up correctly on the gel, so this couldn't be the problem. The DNA should be of high enough concentration, but I'm now running a control gel with higher concentration of only undigested plasmid to check whether I can detect DNA in the samples.
phage434, on 03 October 2012 - 01:57 PM, said:
I agree with Smog187. Likely your gel running is the problem. Does your ladder show up on your gel? How is your gel stained? You should be adding water to 25 ul, if you are using 2.5 ul of a 10x buffer. Also, SmaI wants to be incubated at 25 rather than 37. I'd suggest that you skip the digestion steps until you can see a good band for your undigested DNA when you run a gel.
The other possibility is that you really don't have any DNA in the tube. How was the DNA prepared?
The other possibility is that you really don't have any DNA in the tube. How was the DNA prepared?
The DNA was prepared by miniprep, in the same way that I normally get yields of a few micrograms/ul. Nanodrop confirms this high concentration, but nothing visible on the gel.
Thanks for your suggestion, like I said above I'm now checking higher concentrations of uncut DNA to see whether I can detect the DNA on my gel.
