File:T--Fudan--Agarose gel electrophoresis--crtEB.png

Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.

The first lane was loaded with D2000 DNA ladder whose sizes were marked on the image. We chose Taq DNA polymerase for its low cost and high reliability, and we designed forward and reverse primers for each carotene synthesis enzyme (crt for short). The PCR reaction was composed of 2 μL 10x Taq polymerase buffer, 16 μL H2O, 0.5 μL Taq polymerase, 0.5 μL dNTP (10 mM each), 0.5 μL forward primer (10 mM), 0.1 μL reverse primer (10 mM), and 1 μL bacterial culture or 1μL colony. Using the same forward primer, and different reverse primers, we were able to detect the composition of various crt genes. After PCR, the correct bacterial clones were sent for Sanger sequencing. Once verified, these clones would be used for further experiments.

The sequences of primers are:

> 5-crtE 5-ATGACGGTCTGCGCAAAAAAAC-3

> rev320crtB 5-CCTTCCAGATGATCAAACGCGTAAG-3

> rev320crtE 5-ATGAGAATGAATGGTAGGGCGTC-3

> rev320crtI 5-GGATTAAACTGCTGAATCTGCGCTTC-3

> rev320crtY 5-CCGCGGTATCCATCCACAAG-3