I have cloned my PCR product using the TOPO-TA cloning kit (Life Technologies) and the pCR2.1-TOPO vector.
When I sequenced the different clones, I saw that there were many differences between them and with the sequence of reference. Nearly in each clone appeared one different mutation. I expected to find some differences, because the gene shows CNV polimorphisms, but the variability observed seem to be too high.
Can some of these mutations have been created in the cloning process?
Has anybody had problems with this kit?