Berkeley LBL/Mimi-SchlH

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'''Construction of ''pET3A-(S)-chlH'':'''
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|[[Berkeley_LBL/Project|Project Description]]
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|[[Berkeley_LBL/Methods|Methods]]
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|[[Berkeley_LBL/Notebook|Notebook]]
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|[[Berkeley_LBL/Results|Results and Discussion]]
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|[[Berkeley_LBL/Resources|Resources]]
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|}
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[[Berkeley_LBL/MimiNotebook|Back to Mimi's Notebook]]
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== '''Construction of ''pET3A-(S)-chlH'':'''==
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
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          ''PCR:''
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          1 ul Synechosystis (10ng/ul)
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          10 ul HF Buffer 5x
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          1 ul dNTP
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          5 ul primer mix
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          0.5 ul Phusion
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          32.5 ul H2O
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          --------------
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          50 ul total
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 +
          ''Conditions:''
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          1. 98°C        30s
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          2. 98°C        8s
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          3. 56°C        30s
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          4. 72°C        2:10m
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          5. Go to 2 for additional 29 cycles
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          6. 72°C        10m
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          7. 4°C        --- 
Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
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2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).
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2. Amplification is followed by [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
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4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
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          ''Schl-H''                   
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          41 ul S-chlH fragment
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          5 ul NEB 3 (10x)
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          0.5 ul BSA (100x)
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          1.8 ul NdeI
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          1.2 ul BamHI
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          ------------------
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          50 ul total
Digest in 37°C for 2 hours.
Digest in 37°C for 2 hours.
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6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~4kb).
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~4kb).
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7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
 
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8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
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7. '''pET3A:'''
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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.
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[[Berkeley_LBL/Miniprep|Miniprep]] cultures.
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[[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes ''NdeI'' and ''BamHI'' using the following conditions:
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        42.1 ul pET3A plasmid
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        5 ul NEB 4 (10x)
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        0.5 ul BSA (100x)
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        1.2 ul NdeI
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        1.2 ul BamHI
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        ---------------------
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        50 ul total
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[[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~5kb).
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 +
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8. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
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          4.5 ul pET3A
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          12.5 ul S-chlH
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          2 ul Ligase Buffer
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          1 ul Ligase Enzyme     
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          -------------------
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          20 ul total
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9. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
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          7 ul pET3A-(S)-H ligation
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          73 ul H2O
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          20 ul KCM solution
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          100 ul Chemical Competent Novablue/DH10B cells
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          -----------------------------------------
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          200 ul total
Plate onto LB Agar + Carb plate
Plate onto LB Agar + Carb plate
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9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
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10. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
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10. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
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11. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
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11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:  
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12. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:  
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            20 ul DNA
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          20 ul DNA
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            3 ul NEB 4 (10x)
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          3 ul NEB 4 (10x)
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            1 ul NdeI
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          1 ul NdeI
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            1 ul SpeI
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          1 ul SpeI
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            3 ul BSA (10x)
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          3 ul BSA (10x)
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            2.0 ul H2O   
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          2.0 ul H2O   
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            -------------
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          -------------
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            30 ul total
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          30 ul total
Run gel – look for ~4kb and ~5kb band
Run gel – look for ~4kb and ~5kb band
Save glycerol stocks
Save glycerol stocks
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13. Transformation into ''BL21'' cells via [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]:
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          2 ul pET3A-(S)-H-Novablue
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          100 ul BL21 cells
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          20 ul KCM (5x)
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          78 ul H2O
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          ----------------------------
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          200 ul total
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14. [[Berkeley_LBL/Overexpression| Protein Expression]]
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15. Protein Analysis using [[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]

Latest revision as of 20:15, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Back to Mimi's Notebook


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)-H 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

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