Berkeley LBL/JoyceNotebook

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'''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI'''''
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[[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
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[[Berkeley_LBL/JoyceRhodobacter|'''Contruction of pET3a Derivatives Containing ''bchD'' and ''bchI''''']]
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{| border =
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|-
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!
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!''bchD''  
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!''bchI''
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|-
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[[Berkeley_LBL/JoyceCyano|'''Contruction of pET3a Derivatives Containing ''chlD'' and ''chlI''''']]
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|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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Rhodobacter Genomic DNA      1 ul
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GC Buffer                  10 ul
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dNTP                        1 ul
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Primer Mix                  5 ul
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Water                    27.5 ul
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DMSO                      5.0 ul
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Phusion Polymerase        0.5 ul
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{|border =
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|-
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|''bchD''
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|-
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|1.Initial Denaturation
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|2.Denaturation
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|3.Annealing
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|4.Extension
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|5.Repeat step 2 to 4 for 30 times
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|6.Final Extension
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|
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|-
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|  98 °C
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|  98 °C
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|  52 °C
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|  72 °C
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|
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|  72 °C
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|  4 °C
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|-
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|  30 sec
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|  8 sec
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|  30 sec
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|  60 sec
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|
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|  10 min
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| forever
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|}
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{|border =
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|-
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|''bchI''
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|-
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|1.Initial Denaturation
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|2.Denaturation
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|3.Annealing
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|4.Extension
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|5.Repeat step 2 to 4 for 30 times
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|6.Final Extension
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|
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|-
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|  98 °C
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|  98 °C
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|  62 °C
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|  72 °C
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|
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|  72 °C
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|  4 °C
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|-
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|  30 sec
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|  8 sec
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|  30 sec
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|  32 sec
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|
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|  10 min
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| forever
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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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|-
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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 by [[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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8. Pick colonies and innoculate in 2ml LB + 2ul Carbencilin in 37°C shaker overnight.
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9. Extract plasmid DNA from bacteria by [[Berkeley_LBL/Miniprep|Miniprep].
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10. Confirm that the plasmid DNA contains the right construct by [[Berkeley_LBL/Digestion|Digestion for Miniprepped DNA]] and [[Berkeley_LBL/Digestion2|Analytic Digestion]].
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11. Do [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] to subclone plasmids into BL21, which have been done by [[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]].
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'''Protein Expression'''
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'''Protein Analysis'''
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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

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