Amplified PCR product is clear as 1 band in gel. but Ethanol precipitation or magnetic beads purification of same PCR product resulted in extra unknown band. why?
As per my knowledge and experience, the polymerase enzyme might not be able to amplify the sequences properly which causes repetitive products to show after the purification.
You can increase the extension time in each cycle, that might help. Hassan Rafique
Is the second band of very similar size to the expected band? I wonder if the pcr sequence has a polymorphism and the slightly denaturing conditions of dna binding to the beads or precipitation are creating a heteroduplex dna so there is a bubble forming in the dna where there is a base mismatch and this bubble changes the shape of the amplimer ( well half of it anyway) making a heteroduplex that will run slower ( apparently larger) than the perfectly matched duplex dna. Sequencing will prove this idea.
Could it be a primer dimer. This is very common in PCR products. This happens when you have too many primers as compared to what is required.
Or could your DNA be broken down or slightly denatured while isolation?
Well, the situation is like.. If the actual band is 500 bp.. The after purification, another band appears in sample with almost 100bp less i.e. of 400 bp
In that case I would possibly say that the DNA might be denatured or would have been fragmented. Or another possibility could be that either of the things in your Master Mix could be contaminated with any other DNA sample that was around 400bp?
Mostly I would suggest to optimize the protocol and isolate the DNA again. Hope this helps!
What % of gel you are using for electrophoresis before purification?
May be the band is still there but you cannot see it if the gel is of less %.
Try a gel of 3 or 3.5 % to check if its a one band or two showing as one sharp band. Usually high % of gel give good separation between bands of smaller difference.
Other possibilities could be crosss contamination. Try usingfresh /new reagents to troubleshoot.
Hope this helps
There are several possible reasons why an extra band might appear after PCR product purification:
To determine the cause of the extra band, it may be helpful to run a gel of the unpurified PCR product side-by-side with the purified product. This will help to determine whether the extra band is a result of the purification process or whether it was present in the original PCR product. Additionally, sequencing the purified PCR product can help to confirm whether it is the intended target sequence or something else.
Some literature that may be useful for troubleshooting PCR product purification and analysis include:
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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