Does anyone know, why would the amplification of CHD gene from female Chicken blood DNA gives only one band at 280-290bp in 3% agarose gel instead of two bands at 281bp and 310bp?
I have attempted with different PCR conditions to optimize the amplification of CHD gene, yet only a single band was shown in the 3% agarose gel.
If anybody have had similar experiences or any possible reason, I would appreciate the support.
Thank you!
It can be happened. Could be interesting if you try with different combination like 1.5%, 2%, 2.5% with 10 or 5ul of Ethidium bromide.
Hi, You may try the followings-
1. Reduce the annealing temperature to see if the other band shows up.
2. Use a 2% TAE-Ethidium Bromide gel.
All the best wishes!
Thank you!
And yes, I have tired 1.8% and 2.8% gel as well. However, would it help if I tried 1% gel?
Hi Ruwini,
The percentage of gel depends on the size of the PCR product. The higher the size, the lesser the percentage and vice-versa. You may try 1.5 and 2% simultaneously. Again, best wishes!
Is your size standard separating well on your gels?
Are all your results from the same dna sample and if so can you try another sample in case there is a sample mix up or you have a sample with a polymorphism/deletion on one amplimer under the 3' end of one primer
Is your negative control negative ( no amplification) to rule out contamination from male amplimer taking over the reaction?
what is the OD260/280 ratio of the dna sample?
Are these new primers or have they worked before in your hands?
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