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]]
 +
|[[Berkeley_LBL/Methods|Methods]]
 +
|[[Berkeley_LBL/Notebook|Notebook]]
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|[[Berkeley_LBL/Results|Results and Discussion]]
 +
|[[Berkeley_LBL/Resources|Resources]]
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|}
-
'''''Cyanobacteria - Synechocystis'''''
+
== '''Construction of pET3A Derivatives Containing R-bchH, R-bchI, and R-bchD''' (Simultaneous Ligation) ==
-
        '''S-chlH:''' 3996 base pairs
+
-
        '''S-chlI''': 1074 base pairs
+
-
        '''S-chlD''': 2031 base pairs
+
-
[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides]]
 
-
'''pET3A:'''
+
'''''Rhodobacter sphaeroides'''''
 +
        '''R-bchH:''' 3582 base pairs
 +
        '''R-bchI''': 1005 base pairs
 +
        '''R-bchD''': 1695 base pairs
-
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.
+
[[Berkeley_LBL/MimiSequence2|Sequences and Properties of Oligonucleotides for R-bch Genes]]
-
2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.
+
[[Berkeley_LBL/Mimi_RbchHID|'''Construction of ''pET3A-(R)-bchHID''''']]
 +
- Not Successful
-
3. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes''NdeI'' and ''BamHI''using the following conditions:
+
[[Berkeley_LBL/MimiSequence3|Sequences and Properties of Oligonucleotides for R-bch Genes(NEW)]]
-
        42.1 ul pET3A plasmid
+
[[Berkeley_LBL/Mimi_RbchHID2|'''Construction of ''pET3A-(R)-bchHID''' - 2nd Attempt'']]
-
        5 ul NEB 4 (10x)
+
- Not Successful
-
        0.5 ul BSA (100x)
+
-
        1.2 ul NdeI
+
-
        1.2 ul BamHI
+
-
+
-
'''Construction of ''pET3A-(S)-chlH'':'''
 
-
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
 
 +
== '''Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD''' (Sequential Ligation) ==
-
Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
 
-
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 (~4kb).
+
'''''Cyanobacteria Synechocystis'''''
 +
        '''S-chlH:''' 3996 base pairs
 +
        '''S-chlI''': 1074 base pairs
 +
        '''S-chlD''': 2031 base pairs
-
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
+
[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides for S-chl Genes]]
-
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
+
[[Berkeley_LBL/Mimi-SchlH|'''Construction of ''pET3A-(S)-chlH''''']]
-
 
+
[[Berkeley_LBL/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlHI''''']]
-
Digest in 37°C for 2 hours.
+
-
Add 0.5 ul NdeI and 0.5 ul BamHI.
+
[[Berkeley_LBL/Mimi-SchlD|'''Construction of ''pET3A-(S)-chlHID''''']]
-
Digest in 37°C for additional 30 minutes.
+
-
 
+
-
5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
+
-
 
+
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band.
+
-
 
+
-
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
+
-
 
+
-
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
+
-
 
+
-
 
+
-
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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
+
-
 
+
-
11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:
+
-
 
+
-
            20 ul DNA
+
-
            3 ul NEB 4 (10x)
+
-
            1 ul NdeI
+
-
            1 ul SpeI
+
-
            3 ul BSA (10x)
+
-
            2.0 ul H2O 
+
-
            -------------
+
-
            30 ul total
+
-
 
+
-
Run gel – look for ~4kb and ~5kb band
+
-
 
+
-
Save glycerol stocks
+
-
 
+
-
 
+
-
'''Construction of ''pET3A-(S)-chlHI'':'''
+
-
 
+
-
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
+
-
 
+
-
 
+
-
Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene.
+
-
 
+
-
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 (~1kb).
+
-
 
+
-
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
+
-
 
+
-
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlI'' with ''KpnI'' and ''BglII'' using the following conditions:
+
-
 
+
-
 
+
-
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. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
+
-
 
+
-
6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlH'' with ''KpnI'' and ''BamHI'' using the following conditions:
+
-
 
+
-
 
+
-
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.
+
-
 
+
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions.
+
-
 
+
-
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''"
+
-
 
+
-
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
+
-
 
+
-
 
+
-
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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
+
-
 
+
-
11. [[Berkeley_LBL/Digestion2|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
+
-
 
+
-
 
+
-
 
+
-
'''Construction of ''pET3A-(S)-chlHID'':'''
+
-
 
+
-
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlD'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
+
-
 
+
-
            ''PCR:''
+
-
            1 ul Schl-D
+
-
            10 ul HF Buffer 5x
+
-
            1 ul dNTP
+
-
            5 ul primer mix
+
-
            0.5 ul Phusion
+
-
            32.5 ul H2O
+
-
            --------------
+
-
            50 ul total
+
-
 
+
-
            Conditions:
+
-
            98°C 30s
+
-
            98°C 8s
+
-
            61°C 30s
+
-
            72°C 1:10m
+
-
            Go to 2 for additional 29 cycles
+
-
            72°C 10m
+
-
            4°C         --- 
+
-
 
+
-
 
+
-
Amplification introduces sites ''SpeI-rbs'' and ''NotI-BglII'' into the gene.
+
-
 
+
-
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).
+
-
 
+
-
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
+
-
 
+
-
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlD'' with ''SpeI'' and ''NotI'' using the following conditions:
+
-
 
+
-
''Schl-D Sequential Restriction Digestion:''
+
-
             
+
-
              ''Digestion #1''
+
-
              43 ul Schl-D
+
-
              5 ul NEB 2 (10x)
+
-
              0.5 ul BSA (100x)
+
-
              1.5 ul SpeI
+
-
 
+
-
2 hour digestion in 37°C
+
-
Add 0.5 ul SpeI
+
-
30 min digestion in 37°C
+
-
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
+
-
 
+
-
              ''Digestion #2''
+
-
              43 ul Schl-D
+
-
              5 ul NEB 3 (10x)
+
-
              0.5 ul BSA (100x)
+
-
              1.5 ul NotI
+
-
 
+
-
2 hour digestion in 37°C
+
-
Add 0.5 ul NotI
+
-
30 min digestion in 37°C
+
-
 
+
-
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
+
-
[[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion.
+
-
 
+
-
6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlHI'' with ''SpeI'' and ''NotI'' using the following conditions:
+
-
 
+
-
''Sequential Restriction Digestion for pEt3A-(S)-HI:''
+
-
 
+
-
              ''Digestion #1:''
+
-
              43 ul pEt3A-(S)-HI
+
-
              5 ul NEB 2 (10x)
+
-
              0.5 ul BSA (100x)
+
-
              1.5 ul SpeI
+
-
 
+
-
2 hour digestion in 37°C
+
-
Add 0.5 ul SpeI
+
-
30 min digestion in 37°C
+
-
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
+
-
 
+
-
              ''Digestion #2:''
+
-
              43 ul pEt3A-(S)-HI
+
-
              5 ul NEB 3 (10x)
+
-
              0.5 ul BSA (100x)
+
-
              1.5 ul NotI
+
-
 
+
-
2 hour digestion in 37°C
+
-
Add 0.5 ul NotI
+
-
30 min digestion in 37°C
+
-
 
+
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
+
-
 
+
-
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:
+
-
 
+
-
              12 ul pET3A-(S)-HI
+
-
              4 ul Schl-D
+
-
              2 ul Ligase Buffer
+
-
              1 ul Ligase Enzyme
+
-
              1 ul H2O       
+
-
              -------------------
+
-
              20 ul total
+
-
 
+
-
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
+
-
 
+
-
              7 ul pET3A-(S)-HID 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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
+
-
 
+
-
11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:
+
-
 
+
-
            20 ul DNA
+
-
            3 ul NEB 2 (10x)
+
-
            1.8 ul NotI
+
-
            1 ul SpeI
+
-
            3 ul BSA (10x)
+
-
            1.2 ul H2O 
+
-
            -------------
+
-
            30 ul total
+
-
 
+
-
Run gel – look for ~2kb and ~9kb band
+
-
 
+
-
Save glycerol stocks
+

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