From 2007.igem.org

Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD

Cyanobacteria - Synechocystis

       S-chlH: 3996 base pairs
       S-chlI: 1074 base pairs
       S-chlD: 2031 base pairs

Sequences and Properties of Oligonucleotides

pET3A:

1. Innoculate pET3A single colony from plate into 250 mL flask containing 50 mL LB + 50 ul Carb. Allow to grow in 30°C shaker overnight.

2. Miniprep cultures.

3. Restriction Digestion of plasmid pET3A with enzymesNdeI and BamHIusing the following conditions:

        42.1 ul pET3A plasmid
        5 ul NEB 4 (10x)
        0.5 ul BSA (100x)
        1.2 ul NdeI
        1.2 ul BamHI

pET3A-(S)-chlH:

1. Amplify Synechocystis-Cyanobacteria gene S-chlH by PCR (Using Phusion Polymerase) using the following conditions:

Amplification introduces restriction sites NdeI and KpnI-BamHI into the gene.

2. Amplification is followed byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene.

3. PCR Clean Up/Purification

4. Restriction Digestion of plasmid S-chlH with NdeI and BamHI using the following conditions:

Digest in 37°C for 2 hours. Add 0.5 ul NdeI and 0.5 ul BamHI. Digest in 37°C for additional 30 minutes.

5. DNA Gel Electrophoresis is used to confirm the correct band of ~4kb.

6. Gel Extraction is performed to isolate the correct band.

7. Ligate S-chlH to plasmid pET3A", yielding plasmid "pET3A-(S)-chlH"