I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel extraction kit and Invitrogen PCR Purification kit and compared the results. The first two values are from the Qiagen kit, the third value is the PCR product value, and the 4th and 5th results are from the Invitrogen kit as seen in the image. Based on this, how can I increase the A260/280 value and obtain higher yields of DNA from the gel?
Hello,
Any kind of purification kit (gel DNA or PCR DNA) reduce the actual yield of DNA. From the measurement, it seems that you have enough PCR products.
The DNA obtained from the gel purification kit extremely low and absurd. I would suggest you to load the PCR product in a less spaced lane (not as broad as in the picture). After electrophoresis, check the position of DNA through a UV illuminator and cut off the additional agarose. You should load as little as DNA-containing agarose gel in the column to get maximum yield. Mix the agarose in the DNA-binding buffer until it is melted completely according to the manufacturer's protocol. Also, elute the gel with low volume of distilled water/elution buffer.
PCR purification kits are better than gel purification kits to retain the DNA yield. The DNA obtained from PCR purification kit has much lower A260/A280 value than the accepted value (~1.8). You can (1) try to elute in low volume elution buffer, (2) use sterile distilled water instead of elution buffer, (3) incubate the column for a few minutes before elution, (4) use two-steps elution with half-volume, (5) you can warm up the elution buffer/water to enhance the yield.
All the best!
If your PCR has low yield, you probably need to focus on optimizing the PCR protocol first.
Hello, in my experience when I see these values at 260/280- 2060/230, the basic problem is that your DNA has contamination with organic solvents (mixture of phenols or salts), many times the kits are old and the solutions have precipitates, my recommendation is Before doing your PCR reaction, check the integrity of the DNAN on a 2% agarose gel.
Next to check the DNA integrity continue with the PCR optimizaction. Doing a temperature gradient, use DMSO and increase the MgCl2 concentration, they are some reactives that you can check. But if you don´t have DNA, the problem is simple. greetings
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