52 Sanger Sequencing Sample Preparations

For Sanger sequencing, start by sending out 20 µL of ~100 ng/µL DNA of your LONG amplicon for three sequencing reactions with all three primers 27fwd, 515 fwd, and 1492rev. Prepare 10 µL a 1:10 dilution of your 100 µM primers.

If you do not get clear chromatograms to interpret the sequence, next send out your SHORT amplicon for Sanger sequencing with only primers 515fwd and 1492rev. Prepare 10µL a 1:10 dilution of your 100 µM primers.

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

Adapted from: Identification of unknown bacterial isolates using Sanger sequencing of the 16S rRNA gene | CHMI services.