Berkeley LBL/Mimi-SchlH
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Construction of pET3A-(S)-chlH:
1. Amplify Synechocystis-Cyanobacteria gene S-chlH by PCR (Using Phusion Polymerase) using the following conditions:
PCR:
1 ul Synechosystis (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. 56°C 30s
4. 72°C 2:10m
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.
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 (~4kb).
4. Restriction Digestion of gene S-chlH with NdeI and BamHI using the following conditions:
Schl-H
41 ul S-chlH fragment
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
1.8 ul NdeI
1.2 ul BamHI
------------------
50 ul total
Digest in 37°C for 2 hours.
Add 0.5 ul NdeI and 0.5 ul BamHI.
Digest in 37°C for additional 30 minutes.
5. DNA Gel Electrophoresis is used to confirm the correct band of ~4kb.
6. Gel Extraction is performed to isolate the correct band(~4kb).
7. pET3A:
Innoculate pET3A single colony from plate into 250 mL flask containing 50 mL LB + 50 ul Carb. Allow to grow in 30°C shaker overnight.
Miniprep cultures.
Restriction Digestion of plasmid pET3A with enzymes NdeI and BamHI using the following conditions:
42.1 ul pET3A plasmid
5 ul NEB 4 (10x)
0.5 ul BSA (100x)
1.2 ul NdeI
1.2 ul BamHI
---------------------
50 ul total
Gel Extraction is performed to isolate the correct band(~5kb).
8. Ligate S-chlH to plasmid pET3A", yielding plasmid "pET3A-(S)-chlH"
4.5 ul pET3A
12.5 ul S-chlH
2 ul Ligase Buffer
1 ul Ligase Enzyme
-------------------
20 ul total
9. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:
7 ul pET3A-(S)-H ligation
73 ul H2O
20 ul KCM solution
100 ul Chemical Competent Novablue/DH10B cells
-----------------------------------------
200 ul total
Plate onto LB Agar + Carb plate
10. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
11. Miniprep cultures
12. Analytic Digestion using the following conditions:
20 ul DNA
3 ul NEB 4 (10x)
1 ul NdeI
1 ul SpeI
3 ul BSA (10x)
2.0 ul H2O
-------------
30 ul total
Run gel – look for ~4kb and ~5kb band
Save glycerol stocks
13. Transformation into BL21 cells via KCM Competent Cell Transformation:
2 ul pET3A-(S)-H-Novablue
100 ul BL21 cells
20 ul KCM (5x)
78 ul H2O
----------------------------
200 ul total
15. Protein Analysis using SDS-PAGE
