Berkeley LBL/Mimi-SchlI


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Construction of pET3A-(S)-chlHI:

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

          1 ul Synechocystis (10ng/ul)
          10 ul HF Buffer 5x
          1 ul dNTP
          5 ul primer mix
          0.5 ul Phusion
          32.5 ul H2O
          50 ul total
          1. 98°C        30s
          2. 98°C        8s
          3. 63°C        30s
          4. 72°C        40s
          5. Go to 2 for additional 29 cycles
          6. 72°C        10m
          7. 4°C         ---  

Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII into the gene.

2. Amplification is followed by DNA 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:

          41 ul S-chlI
          5 ul NEB 2 (10x)
          0.5 ul BSA (100x)
          1.5 ul KpnI
          1.5 ul BglII
          50 ul total

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

       Sequential Digestion for pET3A-(S)-H:
       Digestion #1:
       41.5 ul pET3A-(S)-H plasmid
       5 ul NEB 3 (10x)
       0.5 ul BSA (100x)
       3 ul BamHI
       50 ul total

Digest in 37°C for 2 hours.

Add 0.5 ul BglII.

Digest in 37°C for additional 30 minutes.

Clean Up/Purification

       Digestion #2:
       41.5 ul pET3A plasmid (BamHI)
       5 ul NEB 1 (10x)
       0.5 ul BSA (100x)
       3 ul KpnI
       50 ul total

6. Gel Extraction is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).

7. Ligate S-chlI to plasmid pET3A-(S)-chlH", yielding plasmid "pET3A-(S)-chlHI" using the following conditions:

       4.5 ul pET3A-(S)-H
       12.5 ul S-chlI
       2 ul Ligase Buffer
       1 ul Ligase Enzyme
       20 ul total

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

         7 ul pET3A-(S)-HI 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 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