Berkeley LBL/Mimi RbchHID

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[[Berkeley_LBL/MimiNotebook|Back to Mimi's Notebook]]
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== '''Construction of ''pET3A-(R)-bchHID'':'''==
== '''Construction of ''pET3A-(R)-bchHID'':'''==
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             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             55°C        30s
+
             3. 55°C        30s
-
             72°C        1:50m
+
             4. 72°C        1:50m
-
             Go to 2 for additional 29 cycles
+
             5. Go to 2 for additional 29 cycles
-
             72°C        10m
+
             6. 72°C        10m
-
             4°C        ---   
+
             7. 4°C        ---   
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             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             62°C        30s
+
             3. 62°C        30s
-
             72°C        32s
+
             4. 72°C        32s
-
             Go to 2 for additional 29 cycles
+
             5. Go to 2 for additional 29 cycles
-
             72°C        10m
+
             6. 72°C        10m
-
             4°C        ---   
+
             7. 4°C        ---   
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             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        10s
+
             2. 98°C        10s
-
             56°C        30s
+
             3. 56°C        30s
-
             72°C        1:00m
+
             4. 72°C        1:00m
-
             Go to 2 for additional 29 cycles
+
             5. Go to 2 for additional 29 cycles
-
             72°C        10m
+
             6. 72°C        10m
-
             4°C        ---   
+
             7. 4°C        ---   
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*FAIL: GENES NOT SUBCLONED
*FAIL: GENES NOT SUBCLONED
 +
 +
*Attempt with new primers and conditions

Latest revision as of 19:30, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Back to Mimi's Notebook


Contents

Construction of pET3A-(R)-bchHID:

July 10, 2007

1. Amplify Rhodobacter sphaerodides gene R-bchH by PCR (Using Phusion Polymerase) using the following conditions: 

           PCR:
           1 ul Rhodobacter (100ng/ul)
           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. 55°C        30s
           4. 72°C        1:50m
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---


Amplification introduces sites NdeI and KpnI-BamHI into the gene.


July 16, 2007

2. Amplify Rhodobacter sphaerodides gene R-bchI by PCR (Using Phusion Polymerase) using the following conditions: 

           PCR:
           1 ul Rhodobacter (100ng/ul)
           10 ul HF Buffer 5x
           1 ul dNTP
           5 ul primer mix
           0.5 ul Phusion
           2.5 ul DMSO
           30 ul H2O
           --------------
           50 ul total

           Conditions:
           1. 98°C        30s
           2. 98°C        8s
           3. 62°C        30s
           4. 72°C        32s
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---


Amplification introduces sites KpnI and SpeI-NsiI-BglII into the gene.

3. Amplify Rhodobacter sphaerodides gene R-bchD by PCR (Using Phusion Polymerase) using the following conditions: 

           PCR:
           1 ul Rhodobacter (100ng/ul)
           10 ul GC Buffer 5x
           1 ul dNTP
           5 ul primer mix
           0.5 ul Phusion
           5 ul DMSO
           27.5 ul H2O
           --------------
           50 ul total

           Conditions:
           1. 98°C        30s
           2. 98°C        10s
           3. 56°C        30s
           4. 72°C        1:00m
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---


Amplification introduces sites SpeI and BamHI into the gene.


July 18, 2007

4. 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.

3. PCR Clean Up/Purification

4. Restriction Digestion of genes and plasmid using the following conditions: 

           pET3A                     
           43 ul pET3A plasmid
           5 ul NEB 4 (10x)
           0.5 ul BSA (100x)
           1.2 ul NdeI
           1.2 ul BamHI
           ------------------
           50 ul total

           bchH                     
           42.1 ul bchH fragment
           5 ul NEB 1 (10x)
           0.5 ul BSA (100x)
           1.4 ul NdeI
           1.0 ul KpnI
           ------------------
           50 ul total

           bchI                     
           42.1 ul bchI fragment
           5 ul NEB 2 (10x)
           0.5 ul BSA (100x)
           1.4 ul KpnI
           1.0 ul SpeI
           ------------------
           50 ul total

           bchD                     
           42.1 ul bchD fragment
           5 ul NEB 2 (10x)
           0.5 ul BSA (100x)
           1.2 ul SpeI
           1.2 ul BamHI
           ------------------
           50 ul total

2 hour digestion in 37°C

Add 0.5 ul of each enzyme to appropriate digestion

30 min digestion in 37°C


July 19, 2007

5. Gel Extraction is performed to isolate the correct bands for all digestions

6. Ligate R-bchH, R-bchI, and R-bchD to plasmid pET3A", yielding plasmid "pET3A-(R)-bchHID" using the following conditions: 

          5 ul pET3A
          4 ul R-bchH
          4 ul R-bchI
          4 ul R-bchD
          2 ul Ligase Buffer
          1 ul Ligase Enzyme      
          -------------------
          20 ul total


July 23, 2007

7. Transformation into DH10B cells using KCM Competent Cell Transformation using the following conditions: 

          8 ul pET3A-(R)-HID ligation
          72 ul H2O
          20 ul KCM solution
          100 ul Chemical Competent DH10B cells
          -----------------------------------------
          200 ul total

Plate onto LB Agar + Carb plate

8. Transformation into DH10B cells using Electroporation Transformation using the following conditions: 

          40 ul DH10B cells
          2 ul pET3A-(R)-HID ligation

Time: 3:10ms


July 24, 2007

9. Innoculate 5 single colonies of each plate and release into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.


July 25, 2007

12. Miniprep cultures

13. Analytic Digestion using the following conditions: 

          20 ul DNA
          3 ul NEB 3 (10x)
          0.6 ul BamHI
          1 ul NdeI
          3 ul BSA (10x)
          2.4 ul H2O  
          -------------
          30 ul total

Run gel – look for ~5kb and ~7kb band

No correct bands showed up.

14. Send in for sequencing - Nothing found.

  • FAIL: GENES NOT SUBCLONED
  • Attempt with new primers and conditions
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