Team:ETH Zurich/lab/protocols

From 2014.igem.org

(Difference between revisions)
(Multi site directed mutagenesis)
(Multi site directed mutagenesis)
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Method
Method
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1. Design mutagenesis primer(s).
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#Design mutagenesis primer(s). The targeted mutation should be in the middle of the primer. Design your primers (including the mutations) to have a Tm >=78°C.
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The targeted mutation should be in the middle of the primer
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#Purify template plasmid from a dam+ E. coli strain via miniprep.
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Design your primers (including the mutations) to have a Tm >=78°C.
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#Set up mutagenesis PCR mix:
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2. Purify template plasmid from a dam+ E. coli strain via miniprep.
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<br>
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3. Set up mutagenesis PCR mix:
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16 μL Water
16 μL Water
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<br>
1.5 μL DMSO(100%)
1.5 μL DMSO(100%)
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<br>
0.5 μL MgSO4(50 µM)
0.5 μL MgSO4(50 µM)
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<br>
10 μL Phusion HF Buffer
10 μL Phusion HF Buffer
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<br>
5 μL T4 Ligase Buffer
5 μL T4 Ligase Buffer
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<br>
5 μL 10x Taq Ligase Buffer
5 μL 10x Taq Ligase Buffer
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<br>
0.5 μL Each Mutagenesis Primer (at 40 µM)
0.5 μL Each Mutagenesis Primer (at 40 µM)
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<br>
2 μL Reverse Biobrick Primer
2 μL Reverse Biobrick Primer
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2 μL dNTPs
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<br>
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2 μL dNTPs (100mM)
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<br>
2 μL Template
2 μL Template
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<br>
2 μL PNK
2 μL PNK
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<br>
2 μL Taq Ligase
2 μL Taq Ligase
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<br>
1 μL Phusion Polymerase (hotstart)
1 μL Phusion Polymerase (hotstart)
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4. Run PCR
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<br>
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37°C for 30 minutes (this is the kinase step)
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#Run PCR
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95°C for 3 minutes
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*37°C for 30 minutes (this is the kinase step)
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Run the following for 20 cycles:
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*95°C for 3 minutes
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95°C for 1 minute
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*Run the following for 20 cycles:
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55°C for 1 minute
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**95°C for 1 minute
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65°C for 30 sec/kb of plasmid length minimum
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**55°C for 1 minute
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4°C infinite
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**65°C for 30 sec/kb of plasmid length minimum
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5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
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**4°C infinite
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6. Incubate 4-6 hours at 37°C.
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#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
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7. Purify PCR product and elute into 30μL.
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#Incubate 4-6 hours at 37°C.
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8. Transform 3μL purified DNA into highly competent cells.
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#Purify PCR product and elute into 30μL.
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9. Screen the transformants for the desired mutation using restriction digest or sequencing.
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#Transform 3μL purified DNA into highly competent cells.
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#Screen the transformants for the desired mutation using restriction digest or sequencing.
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<br>
Notes
Notes
Because the inital kinase step could lead to non-specific binding, it would be best to use a hot-start polymerase for this procedure.
Because the inital kinase step could lead to non-specific binding, it would be best to use a hot-start polymerase for this procedure.
Any combination of DMSO and MgSO4 has shown significant improvments in product.
Any combination of DMSO and MgSO4 has shown significant improvments in product.
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http://openwetware.org/wiki/Richard_Lab:Site_Directed_Mutagenesis
http://openwetware.org/wiki/Richard_Lab:Site_Directed_Mutagenesis

Revision as of 16:00, 10 October 2014

iGEM ETH Zurich 2014