Richard Lab:Site Directed Mutagenesis

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(Site Directed Mutagenesis)
(Site Directed Mutagenesis)
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==Site Directed Mutagenesis==
==Site Directed Mutagenesis==
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#. Design mutagenesis primers.  
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1. Design mutagenesis primers.  
::*The targeted mutation should be included into both primers.
::*The targeted mutation should be included into both primers.
::*The mutation can be as close as 4 bases from the 5-terminus.
::*The mutation can be as close as 4 bases from the 5-terminus.
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::*Design your primers to have a melting temperature >=78°C.  
::*Design your primers to have a melting temperature >=78°C.  
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#. Purify template plasmid from a dam+ E. coli strain via miniprep.  
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2. Purify template plasmid from a dam+ E. coli strain via miniprep.  
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#. Set up mutagenesis PCR mix  
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3. Set up mutagenesis PCR mix  
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36µl water
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::*36µl water
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10µl 5X Phusion Buffer
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::*10µl 5X Phusion Buffer
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1µl dNTPs (25mM each)
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::*1µl dNTPs (25mM each)
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1µl Primer F
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::*1µl Primer F
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1µl Primer R
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::*1µl Primer R
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0.5µl Template DNA
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::*0.5µl Template DNA
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0.5µl Phusion Polymerase
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::*0.5µl Phusion Polymerase
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4. Run PCR  
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4. Run PCR  
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1. 98°C for 30 secs  
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#98°C for 30 secs  
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2. 98°C for 10 secs  
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#Run the following for 20 cycles
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3. 60°C for 30 min  
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##98°C for 10 secs  
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4. 72°C for 30 sec/kb of plasmid length minimum  
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##60°C for 30 min  
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5. Run PCR for 20 cycles
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##72°C for 30 sec/kb of plasmid length minimum  
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6. 72°C for 5 mins
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#72°C for 5 mins
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7. 4°C  
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#4°C infinite
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Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
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5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
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Incubate 2-3 hours at 37°C.  
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6. Incubate 2-3 hours at 37°C.  
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Purify PCR product.  
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7. Purify PCR product and elute into 30μL.  
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Transform purified DNA into highly competent cells.  
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8. Transform 3μL purified DNA into highly competent cells.  
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Screen the transformants for the desired mutation using restriction digest or sequencing as appropriate. Typically 1/4 or 1/8 colonies are correct.
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9. Screen the transformants for the desired mutation using restriction digest or sequencing

Revision as of 17:22, 25 January 2011

Site Directed Mutagenesis

1. Design mutagenesis primers.

  • The targeted mutation should be included into both primers.
  • The mutation can be as close as 4 bases from the 5-terminus.
  • The mutation should be at least 8 bases from the 3-terminus.
  • At least eight non-overlapping bases should be introduced at the 3-end of each primer.
  • At least one G or C should be at the end of each primer.
  • Design your primers to have a melting temperature >=78°C.

2. Purify template plasmid from a dam+ E. coli strain via miniprep. 3. Set up mutagenesis PCR mix

  • 36µl water
  • 10µl 5X Phusion Buffer
  • 1µl dNTPs (25mM each)
  • 1µl Primer F
  • 1µl Primer R
  • 0.5µl Template DNA
  • 0.5µl Phusion Polymerase

4. Run PCR

  1. 98°C for 30 secs
  2. Run the following for 20 cycles
    1. 98°C for 10 secs
    2. 60°C for 30 min
    3. 72°C for 30 sec/kb of plasmid length minimum
  3. 72°C for 5 mins
  4. 4°C infinite

5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary) 6. Incubate 2-3 hours at 37°C. 7. Purify PCR product and elute into 30μL. 8. Transform 3μL purified DNA into highly competent cells. 9. Screen the transformants for the desired mutation using restriction digest or sequencing

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