Berkeley LBL/Mimi-SchlD

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'''Construction of ''pET3A-(S)-chlHID'':'''
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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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[[Berkeley_LBL/MimiNotebook|Back to Mimi's Notebook]]
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== '''Construction of ''pET3A-(S)-chlHID'':'''==
 +
 
 +
 
 +
== '''October 01, 2007''' ==
 +
 
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlD'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlD'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
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             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             61°C        30s
+
             3. 61°C        30s
-
             72°C        1:10m
+
             4. 72°C        1:10m
-
             Go to 2 for additional 29 cycles
+
             5. Go to 2 for additional 29 cycles
-
             72°C        10m
+
             6. 72°C        10m
-
             4°C        ---   
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             7. 4°C        ---   
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''Schl-D Sequential Restriction Digestion:''
''Schl-D Sequential Restriction Digestion:''
                
                
-
              ''Digestion #1''
+
            ''Digestion #1''
-
              43 ul Schl-D
+
            43 ul Schl-D
-
              5 ul NEB 2 (10x)
+
            5 ul NEB 2 (10x)
-
              0.5 ul BSA (100x)
+
            0.5 ul BSA (100x)
-
              1.5 ul SpeI
+
            1.5 ul SpeI
 +
            ------------------
 +
            50 ul total
2 hour digestion in 37°C
2 hour digestion in 37°C
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[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
-
              ''Digestion #2''
+
            ''Digestion #2''
-
              43 ul Schl-D
+
            43 ul Schl-D
-
              5 ul NEB 3 (10x)
+
            5 ul NEB 3 (10x)
-
              0.5 ul BSA (100x)
+
            0.5 ul BSA (100x)
-
              1.5 ul NotI
+
            1.5 ul NotI
 +
            -----------------
 +
            50 ul total
2 hour digestion in 37°C
2 hour digestion in 37°C
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30 min digestion in 37°C
30 min digestion in 37°C
 +
 +
 +
== '''October 07, 2007''' ==
 +
5. Add a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker
5. Add a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker
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''Sequential Restriction Digestion for pEt3A-(S)-HI:''
''Sequential Restriction Digestion for pEt3A-(S)-HI:''
-
              ''Digestion #1:''
+
            ''Digestion #1:''
-
              43 ul pEt3A-(S)-HI
+
            43 ul pEt3A-(S)-HI
-
              5 ul NEB 2 (10x)
+
            5 ul NEB 2 (10x)
-
              0.5 ul BSA (100x)
+
            0.5 ul BSA (100x)
-
              1.5 ul SpeI
+
            1.5 ul SpeI
 +
            ------------------
 +
            50 ul total
2 hour digestion in 37°C
2 hour digestion in 37°C
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[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
-
              ''Digestion #2:''
+
            ''Digestion #2:''
-
              43 ul pEt3A-(S)-HI
+
            43 ul pEt3A-(S)-HI
-
              5 ul NEB 3 (10x)
+
            5 ul NEB 3 (10x)
-
              0.5 ul BSA (100x)
+
            0.5 ul BSA (100x)
-
              1.5 ul NotI
+
            1.5 ul NotI
 +
            -------------------
 +
            50 ul total
2 hour digestion in 37°C
2 hour digestion in 37°C
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9. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:
9. [[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
+
          12 ul pET3A-(S)-HI
-
              4 ul Schl-D
+
          4 ul Schl-D
-
              2 ul Ligase Buffer
+
          2 ul Ligase Buffer
-
              1 ul Ligase Enzyme
+
          1 ul Ligase Enzyme
-
              1 ul H2O         
+
          1 ul H2O         
-
              -------------------
+
          -------------------
-
              20 ul total
+
          20 ul total
 +
 
 +
 
 +
 
 +
== '''October 08, 2007''' ==
 +
 
10. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
10. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
-
              7 ul pET3A-(S)-HID ligation
+
          7 ul pET3A-(S)-HID ligation
-
              73 ul H2O
+
          73 ul H2O
-
              20 ul KCM solution
+
          20 ul KCM solution
-
              100 ul Chemical Competent Novablue cells
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          100 ul Chemical Competent Novablue cells
-
              -----------------------------------------
+
          -----------------------------------------
-
              200 ul total
+
          200 ul total
Plate onto LB Agar + Carb plate
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
+
== '''October 09, 2007''' ==
-
11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:
 
-
            20 ul DNA
+
11. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
-
            3 ul NEB 2 (10x)
+
 
-
            1.8 ul NotI
+
 
-
            1 ul SpeI
+
 
-
            3 ul BSA (10x)
+
== '''October 10, 2007''' ==
-
            1.2 ul H2O   
+
 
-
            -------------
+
 
-
            30 ul total
+
12. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
 +
 
 +
13. [[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
Run gel – look for ~2kb and ~9kb band
Save glycerol stocks
Save glycerol stocks
 +
 +
 +
== '''October 21, 2007''' ==
 +
 +
 +
14. Transformation of pET3A-(S)-chlHID into ''BL21'' cells via [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]:
 +
 +
          10 ul pET3A-(S)-HID-Novablue
 +
          100 ul BL21 cells
 +
          20 ul KCM (5x)
 +
          70 ul H2O
 +
          ----------------------------
 +
          200 ul total
 +
 +
15. [[Berkeley_LBL/Overexpression|Protein Expression]]
 +
 +
16. Protein Analysis using [[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]

Latest revision as of 19:32, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Back to Mimi's Notebook


Contents

Construction of pET3A-(S)-chlHID:

October 01, 2007

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:
           1. 98°C        30s
           2. 98°C        8s
           3. 61°C        30s
           4. 72°C        1:10m
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---


Amplification introduces sites SpeI-rbs and NotI-BglII into the gene.

2. Amplification is followed by DNA 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
           ------------------
           50 ul total

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
           -----------------
           50 ul total

2 hour digestion in 37°C

Add 0.5 ul NotI

30 min digestion in 37°C


October 07, 2007

5. Add 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
           ------------------
           50 ul total

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
           -------------------
           50 ul total

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


October 08, 2007

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


October 09, 2007

11. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.


October 10, 2007

12. Miniprep cultures

13. 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


October 21, 2007

14. Transformation of pET3A-(S)-chlHID into BL21 cells via KCM Competent Cell Transformation: 

          10 ul pET3A-(S)-HID-Novablue
          100 ul BL21 cells
          20 ul KCM (5x)
          70 ul H2O
          ----------------------------
          200 ul total

15. Protein Expression

16. Protein Analysis using SDS-PAGE

Retrieved from "http://2007.igem.org/wiki/index.php/Berkeley_LBL/Mimi-SchlD"