I need to amplify a gene cDNA using PCR reaction and need to check if it got amplified by Agarose gel electrophoresis method. My amplified cDNA should be around 3kb. What percent of Agarose gel should I make? And how much concentration of DNA is required to be loaded in the gel?
A 3 KB size fragment should be readily resolvable on a standard 1% agarose gel. The amount to load depends on if your product is single or double stranded as that determines the molecular weight. You'll need about 20 ng total mass to have a good chance of seeing your band.
Thankyou for the input.
Also, what is the maximum and minimum quantity of cDNA we should have to visualize on agarose gel?
Dear Manita Raina
In my opinion, 1.5% to 2% agarose gel is the best gradient for cDNA and small wells. Use about 3 to 5 microliters of your cDNA sample and please don't use high voltage to increase the speed of your work. Use proper voltage and give time to it.
Good luck.
Well, you probably don't need to worry about a maximum with cDNA, usually the concern is with the low end. Again, about 20 ng total mass is a standard minimal amount. The volume you add will depend on your sample type (ssDNA or dsDNA) and the concentration.
Here are the steps for the cDNA agarose gel electrophoresis protocol:
Regarding the concentration of DNA to be loaded, you should load around 100-200 ng of DNA for a 3 kb PCR product. To determine the concentration of your PCR product, you can use a spectrophotometer or a fluorometer. Alternatively, you can run an agarose gel with known amounts of DNA and compare the intensity of your PCR product band to the standards.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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