Bands are appearing very, very faint! I presume low DNA in gel, but my DNA concentration is over 1ug/ul. This is after my overnight digestion. I used a MaxiPrep protocol to extract DNA from Bacteria culture. (I inoculated from my glycerol stock and extracted the DNA after a MaxiPrep).
My DNA (pUH-dnvamp2-iGluSnFR) seems to show clear band digestion on the gel and accurate band size. My other plasmid (pUH-iGluSnFR) seems to show very faintly on the gel after digesting the extracted DNA plasmid. I screened my glycerol stocks and with a MiniPrep and found one that showed my genes. I proceeded to do a Maxi Prep with that clone. My concentration for this DNA was 1349.3ng/ul, and the A260/A280 was 1.9. I have repeated this digestion multiple times. Yet, the gel run after the Maxi is very low.
I am about to run a PCR, assuming that any little DNA present will be amplified to confirm my genes of interest. My purpose, in the end, is to utilize the plasmid for virus production. Hence, a high DNA conc. is important for high virus yield.
Any help troubleshooting will be appreciated. (Pictures: first one is Gel after MiniPrep, second one is gel after MaxiPrep
Hmm, if the DNA is digested, it might be that the 1 ug/ul might be misleading: the spectrophotometer (if you measured it by it) might simply read DNA fragments that do not show on the gel's band. If you used a quantitative marker, how much did you get very low bands, below the lowest marker's band?
Yeah, it seems the spectrophotometer is misleading, but I checked it with another machine as well. I ran a PCR, and it was positive; the plasmids are expressing the accurate gene.
Lane 2: pUH-dnVamp2-iGluSnFR treated with F & R Primers of iGluSnFR
Lane 3: pUH-dnVamp2-iGluSnFR treated with F & R Primers of Vamp2
Lane 4: pUH-iGluSnFR (2371.7ng/ul) treated with F & R Primers of iGluSnFR
Lane 5: pUH-iGluSnFR (473.9ng/ul) treated with F & R Primers of iGluSnFR
I wanted to run a DNA Precipitation protocol to remove any residual Ethanol. I am also thinking of running a CleanUp, but I am unsure which procedure to use. So far, this is the status.
Gels are not the most accurate of the tools. Since you are saying that you ran a PCR and it was positive—but you could not see the band on the gel—I assume it is a quantitative PCR. In that case, you could try to run the melting curve analysis. If you get a single peak, then this is like having a single band on the gel, even if you cannot see the band on the gel.
Several kits are available for purifying PCR products even without gel-band cutting. The protocol is already in the kits. Fingers crossed.
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