Berkeley LBL/Mimi RbchHID2

From 2007.igem.org

< Berkeley LBL(Difference between revisions)
 
(One intermediate revision not shown)
Line 8: Line 8:
|[[Berkeley_LBL/Resources|Resources]]
|[[Berkeley_LBL/Resources|Resources]]
|}
|}
 +
 +
 +
[[Berkeley_LBL/MimiNotebook|Back to Mimi's Notebook]]
 +
== '''Construction of ''pET3A-(R)-bchHID using New Oligonucleotides'':'''==
== '''Construction of ''pET3A-(R)-bchHID using New Oligonucleotides'':'''==
Line 33: Line 37:
             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             63°C        30s
+
             3. 63°C        30s
-
             72°C        6:00m
+
             4. 72°C        6:00m
-
             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        ---   
Line 56: Line 60:
             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             65°C        30s
+
             3. 65°C        30s
-
             72°C        4:00m
+
             4. 72°C        4:00m
-
             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        ---   
3. Amplify Rhodobacter sphaerodides gene ''R-bchI'' by [[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]] using the following conditions:
3. Amplify Rhodobacter sphaerodides gene ''R-bchI'' by [[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]] using the following conditions:
Line 82: Line 86:
             Conditions:
             Conditions:
-
             98°C        30s
+
             1. 98°C        30s
-
             98°C        8s
+
             2. 98°C        8s
-
             63°C        30s
+
             3. 63°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        ---   
+
             7. 4°C        ---   
Line 111: Line 115:
           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        ---   
   
   

Latest revision as of 19:31, 26 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Back to Mimi's Notebook


Contents

Construction of pET3A-(R)-bchHID using New Oligonucleotides:

August 01, 2007

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

           PCR:
           1 ul Rhodobacter (100ng/ul)
           5 ul Ex Taq Buffer (10x)
           4 ul dNTP
           5 ul primer mix (29bp primer)
           0.5 ul DMSO
           0.25 ul Ex Taq Polymerase
           34.25 ul H2O
           --------------
           50 ul total
  • Heat primer at 98°C prior to addition to mixture
  • Heat PCR reaction mixture at 98°C prior to addition of Ex Taq Polymerase 
           Conditions:
           1. 98°C        30s
           2. 98°C        8s
           3. 63°C        30s
           4. 72°C        6:00m
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---


2. Use PCR fragment of bchH-29bp-w/DMSO as template, perform new PCR (Using TaKaRa Ex Taq Polymerase) using the following conditions: 

           PCR:
           1 ul bchH-29bp-fragment
           5 ul Ex Taq Buffer (10x)
           4 ul dNTP
           5 ul primer mix
           0.5 ul DMSO
           0.25 ul Ex Taq Polymerase
           34.25 ul H2O
           --------------
           50 ul total

           Conditions:
           1. 98°C        30s
           2. 98°C        8s
           3. 65°C        30s
           4. 72°C        4:00m
           5. Go to 2 for additional 29 cycles
           6. 72°C        10m
           7. 4°C         ---

3. Amplify Rhodobacter sphaerodides gene R-bchI by PCR (Using TaKaRa Ex Taq Polymerase) using the following conditions: 

           PCR:
           1 ul Rhodobacter (100ng/ul)
           5 ul Ex Taq Buffer (10x)
           4 ul dNTP
           5 ul primer mix
           0.5 ul DMSO
           0.25 ul Ex Taq Polymerase
           34.25 ul H2O
           --------------
           50 ul total
  • Heat primer at 98°C prior to addition to mixture
  • Heat PCR reaction mixture at 98°C prior to addition of Ex Taq Polymerase 
           Conditions:
           1. 98°C        30s
           2. 98°C        8s
           3. 63°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 KpnI and SpeI-NsiI-BglII into the gene.

4. Amplify Rhodobacter sphaerodides gene R-bchD by PCR (Using PhusionPolymerase) 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
  • Heat primer at 98°C prior to addition to rxn mixture
  • Heat PCR reaction at 98°C prior to addition of Phusion polymerase 
          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.


August 03, 2007

5. 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.

6. PCR Clean Up/Purification


August 07, 2007

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

           pET3A                    
           42 ul pET3A plasmid
           5 ul NEB 3 (10x)
           0.5 ul BSA (100x)
           1.5 ul NdeI
           1.0 ul BamHI
           ------------------
           50 ul total

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

           bchI                     
           42.0 ul bchI fragment
           5 ul NEB 2 (10x)
           0.5 ul BSA (100x)
           1.5 ul KpnI
           1.0 ul SpeI
           ------------------
           50 ul total
          
           Sequential Digestion for bchD:

           Digestion #1                     
           42.8 ul bchD fragment
           5 ul NEB 3 (10x)
           0.5 ul BSA (100x)
           1.7 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

8. Clean Up/Purification for Digestion #1 of bchD 

           Digestion #2 for bchD
           42.8 ul bchD
           5.0 ul NEB 2 (10x)
           0.5 ul BSA (100x)
           1.7 ul SpeI
           --------------------
           50 ul total


August 08, 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
          6 ul R-bchH
          3 ul R-bchI
          3 ul R-bchD
          2 ul Ligase Buffer
          1 ul Ligase Enzyme      
          -------------------
          20 ul total


August 10, 2007

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

          2 ul pET3A-(R)-HID ligation
          78 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
          2.5 ul pET3A-(R)-HID ligation

Time: 3:80ms

Plate onto LB Agar + Carb plates

Leave all plates overnight at 37°C


August 12, 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.


August 13, 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

Some bands showed up but were a little off.

14. Send in some samples for sequencing - Nothing found.

  • FAIL: GENES NOT SUBCLONED
Retrieved from "http://2007.igem.org/wiki/index.php/Berkeley_LBL/Mimi_RbchHID2"