Berkeley LBL/Mimi RbchHID
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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.
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
