Berkeley LBL/MimiNotebook

From 2007.igem.org

(Difference between revisions)
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[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides]]
[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides]]
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'''pET3A:'''
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[[Berkeley_LBL/Mimi-SchlH|'''Construction of ''pET3A-(S)-chlH''''']]
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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/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlI''''']]
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2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.
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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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        42.1 ul pET3A plasmid
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        5 ul NEB 4 (10x)
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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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        ---------------------
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        50 ul total
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4. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~5kb).
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'''Construction of ''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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Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' 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 (~4kb).
 
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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-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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6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~4kb).
 
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7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
 
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8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
 
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-
 
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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.
 
-
 
-
10. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
 
-
 
-
11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:
 
-
 
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            20 ul DNA
 
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            3 ul NEB 4 (10x)
 
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            1 ul NdeI
 
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            1 ul SpeI
 
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            3 ul BSA (10x)
 
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            2.0 ul H2O 
 
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            -------------
 
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            30 ul total
 
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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.
 
-
 
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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 (~1kb).
 
-
 
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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-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/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 (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).
 
-
 
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7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''" using the following conditions:
 
-
 
-
 
-
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:
 
-
 
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            20 ul DNA
 
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            3 ul NEB 1 (10x)
 
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            1.4 ul SpeI
 
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            0.9 ul KpnI
 
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            3 ul BSA (10x)
 
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            1.7 ul H2O 
 
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            -------------
 
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            30 ul total
 
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Run gel – look for ~1kb and ~9kb band
 
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Save glycerol stocks
 
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'''Construction of ''pET3A-(S)-chlHID'':'''
'''Construction of ''pET3A-(S)-chlHID'':'''

Revision as of 06:23, 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)-chlHID:

1. Amplify Synechocystis-Cyanobacteria gene S-chlD by 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 byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb).

3. PCR Clean Up/Purification

4. 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

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

6. Miniprep cultures and prepare for digestion.

7. 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

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

8. Gel Extraction is performed to isolate the correct bands for both digestions (~2kb and ~10kb).

9. 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

10. Transformation into Novablue cells using 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. Miniprep cultures

11. 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

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