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

Construction of pET3A-(S)-chlH:

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

Amplification introduces 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 (~4kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene 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"

8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:

Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions:

           20 ul DNA
           3 ul NEB 4 (10x)
           1 ul NdeI
           1 ul SpeI
           3 ul BSA (10x)
           2.0 ul H2O   
           -------------
           30 ul total

Run gel – look for ~4kb and ~5kb band

Save glycerol stocks

Construction of pET3A-(S)-chlHI:

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

Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII 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 (~1kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlI with KpnI and BglII using the following conditions:

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

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

6. Restriction Digestion of plasmid pET3A-(S)-schlH with KpnI and BamHI using the following conditions:

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

6. Gel Extraction is performed to isolate the correct bands for both digestions.

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

8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:

Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions:

           20 ul DNA
           3 ul NEB 1 (10x)
           1.4 ul SpeI
           0.9 ul KpnI
           3 ul BSA (10x)
           1.7 ul H2O   
           -------------
           30 ul total

Run gel – look for ~1kb and ~9kb band

Save glycerol stocks

Construction of pET3A-(S)-chlHID:

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

           PCR:
           1 ul Schl-D
           10 ul HF Buffer 5x
           1 ul dNTP
           5 ul primer mix
           0.5 ul Phusion
           32.5 ul H2O
           --------------
           50 ul total

           Conditions:
           98°C	30s
           98°C 	8s
           61°C	30s
           72°C	1:10m
           Go to 2 for additional 29 cycles
           72°C	10m
           4°C	        ---  

Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII 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 (~1kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlI with KpnI and BglII using the following conditions:

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

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

6. Restriction Digestion of plasmid pET3A-(S)-schlH with KpnI and BamHI using the following conditions:

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

6. Gel Extraction is performed to isolate the correct bands for both digestions.

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

8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:

Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions:

           20 ul DNA
           3 ul NEB 1 (10x)
           1.4 ul SpeI
           0.9 ul KpnI
           3 ul BSA (10x)
           1.7 ul H2O   
           -------------
           30 ul total

Run gel – look for ~1kb and ~9kb band

Save glycerol stocks