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:
98°C 30s
98°C 8s
55°C 30s
72°C 1:50m
Go to 2 for additional 29 cycles
72°C 10m
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:
98°C 30s
98°C 8s
62°C 30s
72°C 32s
Go to 2 for additional 29 cycles
72°C 10m
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:
98°C 30s
98°C 10s
56°C 30s
72°C 1:00m
Go to 2 for additional 29 cycles
72°C 10m
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. 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
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
16. Protein Analysis
17. Assay Analysis
