Berkeley LBL/MimiNotebook

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'''Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD'''
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{| border="0" cellspacing="8px" cellpadding="15" width="80%"
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|-
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|[[Berkeley_LBL|Home]]
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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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'''''Cyanobacteria - Synechocystis'''''
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== '''Construction of pET3A Derivatives Containing R-bchH, R-bchI, and R-bchD''' (Simultaneous Ligation) ==
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        '''S-chlH:''' 3996 base pairs
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        '''S-chlI''': 1074 base pairs
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        '''S-chlD''': 2031 base pairs
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[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides]]
 
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'''pET3A:'''
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'''''Rhodobacter sphaeroides'''''
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        '''R-bchH:''' 3582 base pairs
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        '''R-bchI''': 1005 base pairs
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        '''R-bchD''': 1695 base pairs
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1. 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/MimiSequence2|Sequences and Properties of Oligonucleotides for R-bch Genes]]
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2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.
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[[Berkeley_LBL/Mimi_RbchHID|'''Construction of ''pET3A-(R)-bchHID''''']]
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- Not Successful
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3. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes''NdeI'' and ''BamHI''using the following conditions:
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[[Berkeley_LBL/MimiSequence3|Sequences and Properties of Oligonucleotides for R-bch Genes(NEW)]]
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        42.1 ul pET3A plasmid
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[[Berkeley_LBL/Mimi_RbchHID2|'''Construction of ''pET3A-(R)-bchHID''' - 2nd Attempt'']]
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        5 ul NEB 4 (10x)
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- Not Successful
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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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'''pET3A-(S)-chlH:'''
 
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1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
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== '''Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD''' (Sequential Ligation) ==
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Amplification introduces restriction sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
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'''''Cyanobacteria Synechocystis'''''
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        '''S-chlH:''' 3996 base pairs
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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.
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        '''S-chlI''': 1074 base pairs
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        '''S-chlD''': 2031 base pairs
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3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
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4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
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Digest in 37°C for 2 hours.
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Add 0.5 ul NdeI and 0.5 ul BamHI.
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Digest in 37°C for additional 30 minutes.
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5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
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[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides for S-chl Genes]]
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6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band.
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[[Berkeley_LBL/Mimi-SchlH|'''Construction of ''pET3A-(S)-chlH''''']]
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7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
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[[Berkeley_LBL/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlHI''''']]
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[[Berkeley_LBL/Mimi-SchlD|'''Construction of ''pET3A-(S)-chlHID''''']]

Latest revision as of 20:29, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources

Construction of pET3A Derivatives Containing R-bchH, R-bchI, and R-bchD (Simultaneous Ligation)

Rhodobacter sphaeroides 

       R-bchH: 3582 base pairs
       R-bchI: 1005 base pairs
       R-bchD: 1695 base pairs

Sequences and Properties of Oligonucleotides for R-bch Genes

Construction of pET3A-(R)-bchHID - Not Successful

Sequences and Properties of Oligonucleotides for R-bch Genes(NEW)

Construction of pET3A-(R)-bchHID - 2nd Attempt - Not Successful


Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD (Sequential Ligation)

Cyanobacteria Synechocystis 

       S-chlH: 3996 base pairs
       S-chlI: 1074 base pairs
       S-chlD: 2031 base pairs

Sequences and Properties of Oligonucleotides for S-chl Genes

Construction of pET3A-(S)-chlH

Construction of pET3A-(S)-chlHI

Construction of pET3A-(S)-chlHID

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