Why all the electrophoresis with RNA are not good
DNA PCR RNA comtaminated
Mauro, I am not sure what you are asking. If you are running standard PCR then RNA contamination should never be an issue. RNA is not stable at 95°C., which is the temperature you are using to denature your polymerase, so any RNA would not influence your final product at all.
Concerning the gel picture you appended: In order to help you troubleshooting, it would be good to know what you were trying to do in the first place.
hi,
the only problem with rna is when you do not know you have it and assume that all of your measured nucleic acid is DNA so you have very little template so a couple more cycles of pcr may be needed. If you have huge amounts of rna then it can bind some magnesium so adding a little more MG can be a good idea in the pcr
Hi thank you all for your answers , in this laboratory expriment we are tring to identify witch of the DNA samples that we had match with the DNA of the suspect of a crime , so for that we use PCR , and one of the topics that they propose us tu discuss is if the degree of contamination influence or study
1. For a simple amplification, low-amount of RNA contamination should not affect your PCR reaction. We have used very "crude" method (no commercial kit, only simple home-made reagents, no RNase treatment) to isolate plant genomic DNA, and still get good PCR bands we want.
However, your DNA samples that contains inhibitory compounds (e.g., sample preparation reagents, excessive protein) can lead to partial or complete inhibition of downstream PCR. PCR inhibitors originating from the starting material include heparin (>0.15mg/mL), proteins such as hemoglobin (>1mg/mL), polysaccharides, chlorophylls, melanin, humic acids, etc. Contaminants from the nucleic acid extraction phase include SDS (>0.01% w/v), phenol (>0.2% w/v), ethanol (>1%), proteinase K, guanidinium, and sodium acetate (>5mM).
Cited from link: https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html
2. If the first lane in your gel is DNA markers, you may need to optimize your gel running. The DNA markers did not separate well on the gel.
Mauro, it might also help to know which DNA isolation protocol you are using.
Yuan and Paul both gave good points about contaminating agents and the relevance of Magnesium in running a successful reaction. Concerning the appearance of your molecular weight markers, the irregularities you see could either be due to not leaving enough time for your gel to settle (leave it at RT for at least 1h) or due to an unequal mix of your molecular weight marker with dye/H2O. Best of luck.
Although I mentioned above that we used gDNA isolated using a "crude" method for PCR, for certain PCR applications (such as quantitative PCR or qPCR), the use of gDNA template should be as pure as possible. Especially, you are doing PCR for linking suspects to "Criminal events". Extreme care should be taken on your PCR. You don't want to send an innocent person to jail due to an error PCR result.
As for setting your gel to solidify mentioned by Felix, if you don't want to wait too long, after you cast the gel in a gel tray, you can place them in 4C. It will be ready in ~15-20 minutes (small-medium gel). I have been doing so, and the gel images are very nice. Of course, you have to be careful handling the tray to prevent EtBr contamination in the 4C chamber.
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