Select colonies on the agar plate to pick. Culture portion of the colony in LB media with antibiotic and grow in 37 degree. Put the remainder of the colony into a correspondingly numbered PCR tube with 20 µL of reaction mix (as below), and pipetting to complete resuspend the bacteria.

A	B
Component	Volume/ul
10x Taq Buffer	2 uL
10 uM Forward Primer	0.5 uL
10 uM Reverse Primer	0.5 uL
Template DNA (bacteria colony)	added later
dNTP 10 mM mix	0.5 uL
Taq DNA polymerase	0.5 uL
distilled water	16 uL
Total	20 uL

Prepare PCR reaction mix before step 1, and place the mix on ice. Primers used for screening crt genes insertion are:

> 5-crtB
5-ATGAATAATCCGTCGTTACTCAATCATGC-3

> 5-crtE
5-ATGACGGTCTGCGCAAAAAAAC-3

> 5-crtI
5-ATGAAACCAACTACGGTAATTGGTGC-3

> 5-crtY
5-ATGCAACCGCATTATGATCTGATTC-3

> rev320crtB
5-CCTTCCAGATGATCAAACGCGTAAG-3

> rev320crtE
5-ATGAGAATGAATGGTAGGGCGTC-3

> rev320crtI
5-GGATTAAACTGCTGAATCTGCGCTTC-3

> rev320crtY
5-CCGCGGTATCCATCCACAAG-3

A	B
Temperature	Time
94℃	05:00
94℃	00:30
60℃	00:30
72℃	03:30, cycle 3-4-5 for 25 times
72℃	05:00
16℃	∞

The PCR program above is for screening crt genes insertion during 2022 iGEM season.

Agarose gel electrophoresis of PCR products. One lane on the gel is loaded with 6 uL D2000 DNA ladder. Run the gel at 100 V for 20-30 minutes, and examine the gel under UV.

The correct bacterial clones are sent for Sanger sequencing. Once verified, these clones would be used for further experiments.