Trying to clone human TOPORS gene into AAV vector plasmids. The TOPORS gene was cloned from cDNA by PCR and purified. Restriction enzyme digestion was used to isolate existing vector and make it ready for insertion of TOPORS gene. The TOPORS gene, however, was amplified separately by many primers as it is quite large (+2000 bp). The fragments are then purified and ligated together with the AAV vector plasmids and transformed into E.coli.

Since the TOPORS gene is quite lengthy (+2000 bp), it was amplified in sections with multiple primers, and these amplified fragments were then purified. These gene fragments were ligated with the AAV vector plasmids and subsequently introduced into E.coli.

Following the transformation process, E.coli colonies were selected and their plasmid content was isolated. To preliminarily validate successful cloning, I cleaved the extracted plasmids with restriction enzymes located at the borders of the TOPORS gene.

Here's where things got interesting.

In each batch of around 10 colonies, only one or two showed the expected two bands on the gel, corresponding to the right base pair size. The rest showed an unexpected pattern with three bands appearing on the gel, including a faint band in the middle. This suggests the presence of three fragments despite there being only two expected cut sites. In addition, some colonies showed two clear bands, but with an incorrect base pair size. I attempted to use different restriction enzymes, but it did not resolve the issue.

Despite these oddities, a plasmid from one of the colonies showing the expected pattern was sent for sequencing. As the TOPORS gene is large, multiple primers were used for the sequencing. The sequencing results revealed an unexpected deletion of approximately 360 bp from the middle of the TOPORS gene in the ligated plasmid. Aside from this missing segment, the rest of the gene was accurately coded.

Any advice or suggestions would be greatly appreciated.