Berkeley LBL/MimiNotebook
From 2007.igem.org
Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD
Cyanobacteria - Synechocystis
S-chlH: 3996 base pairs
S-chlI: 1074 base pairs
S-chlD: 2031 base pairs
Sequences and Properties of Oligonucleotides
pET3A:
1. 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.
2. Miniprep cultures.
3. Restriction Digestion of plasmid pET3A with enzymesNdeI and BamHIusing 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
Construction of pET3A-(S)-chlH:
1. Amplify Synechocystis-Cyanobacteria gene S-chlH by PCR (Using Phusion Polymerase) using the following conditions:
Amplification introduces sites NdeI and KpnI-BamHI into the gene.
2. Amplification is followed byDNA 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:
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.
7. Ligate S-chlH to plasmid pET3A", yielding plasmid "pET3A-(S)-chlH"
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 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
Construction of pET3A-(S)-chlHI:
1. Amplify Synechocystis-Cyanobacteria gene S-chlI by PCR (Using Phusion Polymerase) using the following conditions:
Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII into the gene.
2. Amplification is followed byDNA 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. DNA Gel Electrophoresis is used to confirm the correct band of ~4kb.
6. 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.
7. Ligate S-chlI to plasmid pET3A-(S)-chlH", yielding plasmid "pET3A-(S)-chlHI"
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
Construction of pET3A-(S)-chlHID:
1. Amplify Synechocystis-Cyanobacteria gene S-chlD by PCR (Using Phusion Polymerase) using the following conditions:
PCR:
1 ul Schl-D
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
61°C 30s
72°C 1:10m
Go to 2 for additional 29 cycles
72°C 10m
4°C ---
Amplification introduces sites SpeI-rbs and NotI-BglII into the gene.
2. Amplification is followed byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb).
4. Restriction Digestion of gene S-chlD with SpeI and NotI using the following conditions:
Schl-D Sequential Restriction Digestion:
Digestion #1
43 ul Schl-D
5 ul NEB 2 (10x)
0.5 ul BSA (100x)
1.5 ul SpeI
2 hour digestion in 37°C Add 0.5 ul SpeI 30 min digestion in 37°C Clean Up/Purification
Digestion #2
43 ul Schl-D
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
1.5 ul NotI
2 hour digestion in 37°C Add 0.5 ul NotI 30 min digestion in 37°C
5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker Miniprep cultures and prepare for digestion.
6. 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
2 hour digestion in 37°C Add 0.5 ul SpeI 30 min digestion in 37°C Clean Up/Purification
Digestion #2:
43 ul pEt3A-(S)-HI
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
1.5 ul NotI
2 hour digestion in 37°C Add 0.5 ul NotI 30 min digestion in 37°C
6. Gel Extraction is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
7. 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
8. 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
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 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
