Berkeley LBL/Mimi-SchlD
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Construction of pET3A-(S)-chlHID:
October 01, 2007
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:
1. 98°C 30s
2. 98°C 8s
3. 61°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 SpeI-rbs and 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 (~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
------------------
50 ul total
2 hour digestion in 37°C
Add 0.5 ul SpeI
30 min digestion in 37°C
Digestion #2
43 ul Schl-D
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
1.5 ul NotI
-----------------
50 ul total
2 hour digestion in 37°C
Add 0.5 ul NotI
30 min digestion in 37°C
October 07, 2007
5. Add a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker
6. Miniprep cultures and prepare for digestion.
7. 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
------------------
50 ul total
2 hour digestion in 37°C
Add 0.5 ul SpeI
30 min digestion in 37°C
Digestion #2:
43 ul pEt3A-(S)-HI
5 ul NEB 3 (10x)
0.5 ul BSA (100x)
1.5 ul NotI
-------------------
50 ul total
2 hour digestion in 37°C
Add 0.5 ul NotI
30 min digestion in 37°C
8. Gel Extraction is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
9. 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
October 08, 2007
10. 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
October 09, 2007
11. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
October 10, 2007
12. Miniprep cultures
13. 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
October 21, 2007
14. Transformation of pET3A-(S)-chlHID into BL21 cells via KCM Competent Cell Transformation:
10 ul pET3A-(S)-HID-Novablue
100 ul BL21 cells
20 ul KCM (5x)
70 ul H2O
----------------------------
200 ul total
16. Protein Analysis using SDS-PAGE
