Berkeley LBL/Mimi-SchlH

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Revision as of 20:03, 26 October 2007

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Construction of pET3A-(S)-chlH:

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

          PCR:
          1 ul Synechosystis (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

          Conditions:
          1. 98°C        30s
          2. 98°C        8s
          3. 56°C        30s
          4. 72°C        2:10m
          5. Go to 2 for additional 29 cycles
          6. 72°C        10m
          7. 4°C         ---  

Amplification introduces sites NdeI and KpnI-BamHI 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 (~4kb).

3. PCR Clean Up/Purification

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

          Schl-H                    
          41 ul S-chlH fragment
          5 ul NEB 3 (10x)
          0.5 ul BSA (100x)
          1.8 ul NdeI
          1.2 ul BamHI
          ------------------
          50 ul total

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(~4kb).

7. pET3A:

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.

Miniprep cultures.

Restriction Digestion of plasmid pET3A with enzymes NdeI and BamHI using 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
        ---------------------
        50 ul total

Gel Extraction is performed to isolate the correct band(~5kb).

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

         4.5 ul pET3A
         12.5 ul S-chlH
         2 ul Ligase Buffer
         1 ul Ligase Enzyme      
         -------------------
         20 ul total

9. 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/DH10B cells
         -----------------------------------------
         200 ul total

Plate onto LB Agar + Carb plate

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

11. Miniprep cultures

12. 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

13. Transformation into BL21 cells via KCM Competent Cell Transformation:

         2 ul pET3A-(S)-H-Novablue
         100 ul BL21 cells
         20 ul KCM (5x)
         78 ul H2O
         ----------------------------
         200 ul total

14. Protein Expression

15. Protein Analysis using SDS-PAGE