Berkeley LBL/Mimi RbchHID2
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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.
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
