Berkeley LBL/Mimi-SchlI
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1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions: | 1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions: | ||
| + | ''PCR:'' | ||
| + | 1 ul Synechocystis (10ng/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. 63°C 30s | ||
| + | 4. 72°C 40s | ||
| + | 5. Go to 2 for additional 29 cycles | ||
| + | 6. 72°C 10m | ||
| + | 7. 4°C --- | ||
Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene. | Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene. | ||
Revision as of 19:57, 26 October 2007
| Home | Project Description | Methods | Notebook | Results and Discussion | Resources |
Construction of pET3A-(S)-chlHI:
1. Amplify Synechocystis-Cyanobacteria gene S-chlI by PCR (Using Phusion Polymerase) using the following conditions:
PCR:
1 ul Synechocystis (10ng/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. 63°C 30s
4. 72°C 40s
5. Go to 2 for additional 29 cycles
6. 72°C 10m
7. 4°C ---
Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII into the gene.
2. 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 (~1kb).
4. Restriction Digestion of gene S-chlI with KpnI and BglII using the following conditions:
Digest in 37°C for 2 hours.
Add 0.5 ul KpnI and 0.5 ul BglII.
Digest in 37°C for additional 30 minutes.
5. Restriction Digestion of plasmid pET3A-(S)-schlH with KpnI and BamHI using the following conditions:
Digest in 37°C for 2 hours.
Add 0.5 ul KpnI and 0.5 ul BglII.
Digest in 37°C for additional 30 minutes.
6. Gel Extraction is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).
7. Ligate S-chlI to plasmid pET3A-(S)-chlH", yielding plasmid "pET3A-(S)-chlHI" using the following conditions:
8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:
Plate onto LB Agar + Carb plate
9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
10. Miniprep cultures
11. Analytic Digestion using the following conditions:
20 ul DNA
3 ul NEB 1 (10x)
1.4 ul SpeI
0.9 ul KpnI
3 ul BSA (10x)
1.7 ul H2O
-------------
30 ul total
Run gel – look for ~1kb and ~9kb band
Save glycerol stocks
12. Transformation into BL21 cells via KCM Competent Cell Transformation:
