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| - | '''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI''''' | + | [[User:Joyxi|'''Joyce's user page]] |
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| - | 1. Amplify Rhodobacter's gene and introduce restriction sites by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]], which allow cloning PCR fragments into pET3a
| + | [[Berkeley_LBL/JoyceRhodobacter|'''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI''''']] |
| - | {| border =
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| - | !
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| - | !''bchD''
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| - | !''bchI''
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| + | [[Berkeley_LBL/JoyceCyano|'''Contruction of pET3a Derivatives Containing ''chlD'' and ''chlI''''']] |
| - | |length of gene fragment
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| - | |1695 b.p.
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| - | |1005 b.p.
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| - | |Restriction sites introduced when amplified by PCR
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| - | |NdeI, BamHI
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| - | |NdeI, BglI
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| - | |}
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| - | 2. Do [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] to confirm that the length of the PCR fragments are the same as the corresponding target gene fragments.
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| - | 3. Remove any leftover PCR enzyme in the samples by [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]].
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| - | 4. [[Berkeley_LBL/Digestion|Digestion for PCR Products]] using specific restriction enzymes
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| - | {| border =
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| - | !''bchD''
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| - | |NdeI, BamHI
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| - | |-
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| - | !''bchI''
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| - | |NdeI, BglI
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| - | |}
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| - | 4. Digested ''bchD'' and ''bchI'' are subjected to [[Berkeley_LBL/GelExtraction|Gel Extraction]] to ensure that only pure, digested DNA is obtained before doing ligation.
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| - | 5. Ligate digested and purified PCR fragments with digested pET3a[[Berkeley_LBL/Ligation|Ligation]] yielding plasmids of pETBCHD and pETBCHI.
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| - | 6. Use small portion of the plasmids to do [[Berkeley_LBL/Digestion2|Analytic Digestion]]
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| - | 7. Do [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] to subclone plasmids into DH10B competent cells, which have been done by [[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]].
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Latest revision as of 02:35, 27 October 2007
Joyce's user page
Contruction of pET3a Derivatives Containing bchD and bchI
Contruction of pET3a Derivatives Containing chlD and chlI
