PCR and Agarose Gel Electrophoresis

April 9, 2016

 

As starting a lab in 2016, I tried not to install the EtBr system in the lab. Instead, I chose a non-EtBr dye and the blue-green LED system. The dye is not as harmful as EtBr, and the detection system is based on an LED, not UV. Therefore, I do not have to worry about the gel or buffer waste. Moreover, I can just casually open the door of the device to cut out DNA bands, as the system does not use the UV light, although I have to wear orange glasses to see bands. I hope this investment is worthwhile. By the way, the name of the dye is "Midori-Green". "Midori" means "Green" in Japanese, so "Midori-Green" is "Green-Green".

I also started a kind of cloning by PCR. However, I kept having the same band no matter what I tried to amplify by PCR. When you are starting a lab, you cannot easily tell what is wrong, because everything is new!! It had been tough, but after a week of struggle, I finally found that just a mixture of the Phusion buffer and Takara DNA loading dye gives a strong signal at around 500 bp, which completely killed my PCR. This signal is strong until after a 15 min run, but disappears for some reason after about a 30 min run. Thanks to the Dr. Shibasaki lab people, I was able to analyze my PCR samples with EtBr (picture). The lower bands are my PCR products, but the upper ones are from the buffer mixture. This non-specific band is more reddish than the real DNA bands, suggesting that this is a sort of chemical reaction (not DNA-EtBr interaction).
I switched to a different loading dye, and now have good PCR amplifications. It was a tough trouble shooting, but finally done. Be careful with the combination of the Phusion buffer and Takara dye.

Phusion bufferとTakara DNA loading dyeを混ぜると、なぜか強烈なバンドが現れました。気付くのに苦労しましたが、これでやっとPCRを使った実験を進められそうです。

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