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

Construction of pET3A-(S)-chlH

Construction of pET3A-(S)-chlI

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 SpeI-rbs and 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 (~2kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlD with SpeI and NotI using the following conditions:

Schl-D Sequential Restriction Digestion:

              Digestion #1
              43 ul Schl-D
              5 ul NEB 2 (10x)
              0.5 ul BSA (100x)
              1.5 ul SpeI

2 hour digestion in 37°C

Add 0.5 ul SpeI

30 min digestion in 37°C

Clean Up/Purification

              Digestion #2
              43 ul Schl-D
              5 ul NEB 3 (10x)
              0.5 ul BSA (100x)
              1.5 ul NotI

2 hour digestion in 37°C

Add 0.5 ul NotI

30 min digestion in 37°C

5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker

6. Miniprep cultures and prepare for digestion.

7. Restriction Digestion of plasmid pET3A-(S)-schlHI with SpeI and NotI using the following conditions:

Sequential Restriction Digestion for pEt3A-(S)-HI:

              Digestion #1:
              43 ul pEt3A-(S)-HI
              5 ul NEB 2 (10x)
              0.5 ul BSA (100x)
              1.5 ul SpeI

2 hour digestion in 37°C

Add 0.5 ul SpeI

30 min digestion in 37°C

Clean Up/Purification

              Digestion #2:
              43 ul pEt3A-(S)-HI
              5 ul NEB 3 (10x)
              0.5 ul BSA (100x)
              1.5 ul NotI

2 hour digestion in 37°C

Add 0.5 ul NotI

30 min digestion in 37°C

8. Gel Extraction is performed to isolate the correct bands for both digestions (~2kb and ~10kb).

9. Ligate S-chlD to plasmid pET3A-(S)-chlHI", yielding plasmid "pET3A-(S)-chlHID" using the following conditions:

             12 ul pET3A-(S)-HI
             4 ul Schl-D
             2 ul Ligase Buffer
             1 ul Ligase Enzyme
             1 ul H2O        
             -------------------
             20 ul total

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

             7 ul pET3A-(S)-HID ligation
             73 ul H2O
             20 ul KCM solution
             100 ul Chemical Competent Novablue cells
             -----------------------------------------
             200 ul total

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 2 (10x)
           1.8 ul NotI
           1 ul SpeI
           3 ul BSA (10x)
           1.2 ul H2O  
           -------------
           30 ul total

Run gel – look for ~2kb and ~9kb band

Save glycerol stocks