Berkeley LBL/Mimi-SchlI

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|[[Berkeley_LBL/Resources|Resources]]
|[[Berkeley_LBL/Resources|Resources]]
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[[Berkeley_LBL/MimiNotebook|Back to Mimi's Notebook]]
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== '''Construction of ''pET3A-(S)-chlHI'':''' ==
== '''Construction of ''pET3A-(S)-chlHI'':''' ==
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1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
 +
          ''PCR:''
 +
          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
 +
          --------------
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          50 ul total
 +
 +
          ''Conditions:''
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          1. 98°C        30s
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          2. 98°C        8s
 +
          3. 63°C        30s
 +
          4. 72°C        40s
 +
          5. Go to 2 for additional 29 cycles
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          6. 72°C        10m
 +
          7. 4°C        --- 
Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene.
Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene.
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4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlI'' with ''KpnI'' and ''BglII'' using the following conditions:
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlI'' with ''KpnI'' and ''BglII'' using the following conditions:
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          ''S-chlI:''
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          41 ul S-chlI
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          5 ul NEB 2 (10x)
 +
          0.5 ul BSA (100x)
 +
          1.5 ul KpnI
 +
          1.5 ul BglII
 +
          ------------------
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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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Digest in 37°C for additional 30 minutes.
Digest in 37°C for additional 30 minutes.
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5. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlH'' with ''KpnI'' and ''BamHI'' using the following conditions:
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5. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-H'' with ''KpnI'' and ''BamHI'' using the following conditions:
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        Sequential Digestion for pET3A-(S)-H:
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        ''Digestion #1'':
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        41.5 ul pET3A-(S)-H plasmid
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        5 ul NEB 3 (10x)
 +
        0.5 ul BSA (100x)
 +
        3 ul BamHI
 +
        ---------------------
 +
        50 ul total
Digest in 37°C for 2 hours.
Digest in 37°C for 2 hours.
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Add 0.5 ul KpnI and 0.5 ul BglII.
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Add 0.5 ul BglII.
Digest in 37°C for additional 30 minutes.
Digest in 37°C for additional 30 minutes.
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 +
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
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        ''Digestion #2'':
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        41.5 ul pET3A plasmid (BamHI)
 +
        5 ul NEB 1 (10x)
 +
        0.5 ul BSA (100x)
 +
        3 ul KpnI
 +
        ---------------------
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        50 ul total
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).
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7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''" using the following conditions:
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''" using the following conditions:
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        4.5 ul pET3A-(S)-H
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        12.5 ul S-chlI
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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
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
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          7 ul pET3A-(S)-HI 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 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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Save glycerol stocks
Save glycerol stocks
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12. Transformation into ''BL21'' cells via [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]:
 
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13. [[Berkeley_LBL/Overexpression| Protein Expression]]
 
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14. Protein Analysis using [[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]
 

Latest revision as of 20:13, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Back to Mimi's Notebook


Construction of pET3A-(S)-chlHI:

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

          PCR:
          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

          Conditions:
          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: 

          S-chlI:
          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

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