Contamination in sequencing sample

[Danielsuarez1609]

Hi,

I always get a comment that my sequencing samples (T vector clone) are contaminated. Can anyone tell me what does that mean?

Thanks in Advance.

[ARGERINE (Dr. Gaganjot Singh)]

@Danielsuarez1609

I always get a comment that my sequencing samples (T vector clone) are contaminated. Can anyone tell me what does that mean?

Daniel Suarez

I was going through some old unanswered posts and found yours. I hope that you’ve already circumvented the issue.

The most common issue is that Sanger sequencing requires one clean plasmid species + one primer. If the sample contains more than one plasmid sequence, the chromatogram will show overlapping peaks, especially at the beginning. This often happens if you pick a mixed colony, have vector-only plus insert-containing plasmids together, leftover PCR product/genomic DNA, or contaminated primers.

How to fix the issue : Re-streak and pick a single isolated colony, confirm the insert by colony PCR, make a clean miniprep, and resend for sequencing.