Berkeley LBL/MimiNotebook

From 2007.igem.org

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[[Berkeley_LBL/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlI''''']]
[[Berkeley_LBL/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlI''''']]
   
   
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[[Berkeley_LBL/Mimi-SchlD|'''Construction of ''pET3A-(S)-chlD''''']]
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'''Construction of ''pET3A-(S)-chlHID'':'''
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1. Amplify Synechocystis-Cyanobacteria gene ''S-chlD'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
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            ''PCR:''
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            1 ul Schl-D
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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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            98°C 30s
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            98°C 8s
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            61°C 30s
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            72°C 1:10m
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            Go to 2 for additional 29 cycles
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            72°C 10m
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            4°C         --- 
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Amplification introduces sites ''SpeI-rbs'' and ''NotI-BglII'' 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 (~2kb).
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3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
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4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlD'' with ''SpeI'' and ''NotI'' using the following conditions:
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''Schl-D Sequential Restriction Digestion:''
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              ''Digestion #1''
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              43 ul Schl-D
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              5 ul NEB 2 (10x)
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              0.5 ul BSA (100x)
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              1.5 ul SpeI
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2 hour digestion in 37°C
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Add 0.5 ul SpeI
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30 min digestion in 37°C
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[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
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              ''Digestion #2''
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              43 ul Schl-D
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              5 ul NEB 3 (10x)
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              0.5 ul BSA (100x)
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              1.5 ul NotI
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2 hour digestion in 37°C
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Add 0.5 ul NotI
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30 min digestion in 37°C
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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
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6. [[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion.
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7. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlHI'' with ''SpeI'' and ''NotI'' using the following conditions:
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''Sequential Restriction Digestion for pEt3A-(S)-HI:''
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              ''Digestion #1:''
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              43 ul pEt3A-(S)-HI
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              5 ul NEB 2 (10x)
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              0.5 ul BSA (100x)
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              1.5 ul SpeI
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2 hour digestion in 37°C
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Add 0.5 ul SpeI
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30 min digestion in 37°C
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[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
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              ''Digestion #2:''
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              43 ul pEt3A-(S)-HI
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              5 ul NEB 3 (10x)
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              0.5 ul BSA (100x)
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              1.5 ul NotI
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2 hour digestion in 37°C
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Add 0.5 ul NotI
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30 min digestion in 37°C
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8. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
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9. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:
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              12 ul pET3A-(S)-HI
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              4 ul Schl-D
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              2 ul Ligase Buffer
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              1 ul Ligase Enzyme
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              1 ul H2O       
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              -------------------
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              20 ul total
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10. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
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              7 ul pET3A-(S)-HID 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
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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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
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11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:
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            20 ul DNA
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            3 ul NEB 2 (10x)
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            1.8 ul NotI
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            1 ul SpeI
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            3 ul BSA (10x)
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            1.2 ul H2O 
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            -------------
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            30 ul total
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Run gel – look for ~2kb and ~9kb band
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Save glycerol stocks
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Revision as of 06:31, 26 October 2007

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)-chlD

Retrieved from "http://2007.igem.org/wiki/index.php/Berkeley_LBL/MimiNotebook"