Hello everyone!
I’m trying to purify from gel a PCR product (about 1300 bp image A) that I should clone in pJet1.2 for sequencing. After cutting the band, I purified with the QIAquick Gel Extraction kit following the protocol.
Loading the purified product on the gel (B), as you can see, are present also other bands (Even those I didn’t cut!)
I tried to repeat the purification several times but the result doesn’t change.
Has anyone ever had this? What could be the cause?
Thanks
The presence of non-specific bands after gel purification can happen due to a few reasons:
Incomplete excision of the target band: If the gel slice contains remnants of other bands, those can carry through the purification process.
Contamination: Tools or reagents used may introduce DNA fragments from other reactions.
Overloaded gel: Too much DNA can lead to smearing or non-specific bands being less visible before purification.
Inefficient purification: Residual DNA from gel or improper washing steps can allow non-specific fragments to remain.
Primer-dimers or by-products: These could be co-purified if they are close in size.
Double-check excision accuracy, purification protocol, and reagents.
Thank you for your answer! Unfortunately, I've already implemented all these suggestions but the result doesn't change
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