Richard Lab:Site Directed Mutagenesis

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(New page: ==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...)
(Site Directed Mutagenesis)
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==Site Directed Mutagenesis==
==Site Directed Mutagenesis==
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1. Design mutagenesis primers.  
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#. 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.
Line 9: Line 9:
::*Design your primers to have a melting temperature >=78°C.  
::*Design your primers to have a melting temperature >=78°C.  
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2. Purify template plasmid from a dam+ E. coli strain via miniprep.  
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#. Purify template plasmid from a dam+ E. coli strain via miniprep.  
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3. Set up mutagenesis PCR mix  
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#. Set up mutagenesis PCR mix  
36µl water
36µl water
10µl 5X Phusion Buffer
10µl 5X Phusion Buffer

Revision as of 16:12, 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.
  1. . Purify template plasmid from a dam+ E. coli strain via miniprep.
  2. . 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. 98°C for 10 secs 3. 60°C for 30 min 4. 72°C for 30 sec/kb of plasmid length minimum 5. Run PCR for 20 cycles 6. 72°C for 5 mins 7. 4°C Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage). Incubate 2-3 hours at 37°C. Purify PCR product. Transform purified DNA into highly competent cells. 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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