I had tissue specific staining in some of my embryos where stained structures are evident, such as the somite boundary in the picture included. I had other probes such as heart specific antisense probes that stained ONLY the heart tissue with no background but the sense probe control stained right away and all over. All probes were made from a PCR product with a T7 promoter on the sense strand and an SP6 promoter on the reverse. SP6 probes were clean when nano dropped with a 260/280 at or just above 2. T7 probes were a little less clean with a 260/280 at 1.88-2. I'm not sure I understand why this in situ did not work (the sense probes stained when they should not have). Any advice would be much appreciated.
Hypothesis 1. What did you use as a template to make the PCR product and how did you purify it (e.g. PCR purification kit, band gel extraction)? Could you have a contaminant in the PCR product which was used to make the probes giving the non-specific staining? If the template to make the PCR product was derived from a cDNA library and the PCR product was purified using a PCR purification kit without careful gel analysis on whether there were non-specific amplified products this could be an issue and give rise to unspecific probes with unexpected signal patterns. Hypothesis 2. An ubiquitous signal is common if a colorimetric reaction is carried out for too long. Hypothesis 3. If you used a plasmid as a template to make the PCR product did you design the primers for the PCR correctly, for example are you introducing the T7 promoter within the primers and does the plasmid already have a T7 promoter in a different region than the one targeted for amplification? These are the potential issues that could explain that result, that I can think of, without knowing any particulars of the experimental design. The fish look beautiful though, such a nice model system.
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