Berkeley LBL/Mimi-SchlI
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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:
S-chlI:
41 ul S-chlI
5 ul NEB 2 (10x)
0.5 ul BSA (100x)
1.5 ul KpnI
1.5 ul BglII
------------------
50 ul total
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)-H with KpnI and BamHI using the following conditions:
Sequential Digestion for pET3A-(S)-H:
Digestion #1:
41.5 ul pET3A-(S)-H plasmid
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
3 ul BamHI
---------------------
50 ul total
Digest in 37°C for 2 hours.
Add 0.5 ul BglII.
Digest in 37°C for additional 30 minutes.
Digestion #2:
41.5 ul pET3A plasmid (BamHI)
5 ul NEB 1 (10x)
0.5 ul BSA (100x)
3 ul KpnI
---------------------
50 ul total
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:
4.5 ul pET3A-(S)-H
12.5 ul S-chlI
2 ul Ligase Buffer
1 ul Ligase Enzyme
--------------------
20 ul total
8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:
7 ul pET3A-(S)-HI ligation
73 ul H2O
20 ul KCM solution
100 ul Chemical Competent Novablue cells
-----------------------------------------
200 ul total
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
