I have a series of serial dilutions for a Positive Control I cloned using TOPO TA cloning kit (thermofisher) and competent Ecoli. I wanted to quantify them using Biospec Nano, but all the values are negative - concentration, OD 260/280, etc.
Am I using the wrong spectrophotometer or template? How is PCP measured besided PCR?
Hi Sahar,
there are two possibilities:
- when you quantify nucleic acids by UV absorption you need to properly blank your instrument. I imagine you have been doing this using the same medium (water, TE) in which you resuspended your plasmid, but often with these microliter instruments air bubbles get in the light path and air (double bonds in nitrogen) absorb significantly in the 260-280 nm range. So, make sure you blank without any air bubbles, which means repeat your measurements a few times to be sure of it.
- other option, there is no guarantee when you make a plasmid prep that you extract any plasmid. You might have had a contamination in your culture so that your clone was overgrown by something else, or you might have lost your plasmid during the prep: Load 1-5 ul of your prep on an agarose gel and verify that you have something in your tube. It could also be that the yield was very low, so when you make serial dilutions you go below the sensitivity of your instrument (which is probably around 10-20 ng/ul).
@pietro is right it is essential to blank and measure in exactly the same buffer otherwise the buffer may have a higher absorbance on its own than the dna in its elution buffer so blank using elution buffer in column purifications and with dilution buffer when doing dilutions
Thank you all. @pietro, I believe low copy numbers might be the issue here because the curves in qPCR look great and show that I have isolated my plasmid. @Paul, I will blank using the elution buffer, thank you. @Utku, device calibration is what hadn't occurred to me. Will definitely try that and let you all know what was the problem.
If blanking does not resolve the problem try cleaning the reading head optics of the machine with 0.5M HCL to get rid of dried dna and protein as these machines do easily get dirty and that dirt absorbs in the UV range.
Hello Sahar,
First quantify a known DNA (like company provided pUC18/19 or any other even TA cloning vector provided will do), this will give you the understanding that machine is working well and optical path is clear. You should well consider the above answers but it seems that your DNA prep is having some DNAse contamination and hence your RNA yield is higher than DNA yield which is giving 260/280 a negative value (degradation product). Hence, you should consider autoclaving the TAE buffer and use molecular biology reagents which are DNAse RNAse free. I think this will take care of your problem. Also pay attention to your preparations and handling keeping in mind that you dont want to use contaminated tips and tubes. All the best. If still it is giving the problem than the plasmid is not there in cells and some mutation in genome has made the bacteria resistant to antibiotic. Think carefully and move accordingly.
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