Chromatogram Analysis of 16S Gene after Sanger Sequencing

This video will help you:
1) understand the Sanger sequencing using labeled ddNTPs after PCR
2) assess the quality of a sequence read by using the chromatogram
3) compare your DNA sequence to previously annotated DNA submissions using BLAST
4) identify the potential genus of an unknown isolate using the 16S rRNA gene
We used F27 (5'-AGAGTTTGATC(A/C)TGGCTCAG-3') and (R1492 5'-TACGG(C/T)TACCTTGTTACGACTT-3') for PCR from Heuer et al 1997 (pubmed.ncbi.nl...)
As an advanced option, you can manually edit any incorrectly coded bases. Here is a good list of chromatogram viewers: eurofinsgenomi...
The BLAST website is blast.ncbi.nlm...
Cheers!
-Dr. Drew Rholl (professor and big fan of microbiology)