Berkeley LBL/Mimi-SchlD
From 2007.igem.org
KonniamChan (Talk | contribs) |
KonniamChan (Talk | contribs) |
||
| Line 14: | Line 14: | ||
Conditions: | Conditions: | ||
| - | 98°C 30s | + | 98°C 30s |
| - | 98°C | + | 98°C 8s |
| - | 61°C 30s | + | 61°C 30s |
| - | 72°C 1:10m | + | 72°C 1:10m |
Go to 2 for additional 29 cycles | Go to 2 for additional 29 cycles | ||
| - | 72°C 10m | + | 72°C 10m |
| - | 4°C --- | + | 4°C --- |
Amplification introduces sites ''SpeI-rbs'' and ''NotI-BglII'' into the gene. | Amplification introduces sites ''SpeI-rbs'' and ''NotI-BglII'' into the gene. | ||
| - | 2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb). | + | 2. Amplification is followed by [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb). |
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]] | 3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]] | ||
| Line 59: | Line 59: | ||
30 min digestion in 37°C | 30 min digestion in 37°C | ||
| - | 5. | + | 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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion. | 6. [[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion. | ||
Revision as of 06:28, 26 October 2007
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 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
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
2 hour digestion in 37°C
Add 0.5 ul NotI
30 min digestion in 37°C
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
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
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
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
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
