Knight:Site-directed mutagenesis/Multi site

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==Mutagenesis PCR mix==
==Mutagenesis PCR mix==
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*5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
+
*2.5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
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*X μL (5–50 ng) of dsDNA template
+
*X μL (50 ng) of dsDNA template
-
*X μL (125 ng) of each oligonucleotide primer
+
*X μL of each oligonucleotide primer
 +
**For 1-3 primers, add 100 ng each primer.  For 4-5 primers, add 50 ng each primer.
**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts.  See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts.  See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
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*ddH<sub>2</sub>O to a final volume of 50 &mu;L
+
*ddH<sub>2</sub>O to a final volume of 25 &mu;L
Then add
Then add
*1 &mu;L of PfuTurbo DNA polymerase (2.5 U/&mu;L)
*1 &mu;L of PfuTurbo DNA polymerase (2.5 U/&mu;L)
 +
*1 &mu;L of Taq Ligase?
==Procedure==
==Procedure==
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#Set up mutagenesis PCR mix as described above.
#Set up mutagenesis PCR mix as described above.
#Run PCR
#Run PCR
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##95&deg;C for 2 mins
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##95&deg;C for 1 min
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##95&deg;C for 30 secs
+
##95&deg;C for 1 min
##55&deg;C for 1 min
##55&deg;C for 1 min
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##68&deg;C for 1 min/kb of plasmid length minimum
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##65&deg;C for 2 min/kb of plasmid length minimum
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##Run PCR for 18 cycles for point mutations (even though the Stratagene manual recommends 12, I've found 18 to be preferable).
+
##Run reaction for 30 cycles.
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##68&deg;C for 20 mins
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#*Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
#*Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
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#*Note that it is pretty common to not be able to visualize the PCR product on a gel but yet have the mutagenesis still work.
 
#Cool the reaction to <=37&deg;C
#Cool the reaction to <=37&deg;C
#Add 1&mu;L DpnI restriction enzyme to the PCR tube directly.  (Purification is not necessary at this stage).
#Add 1&mu;L DpnI restriction enzyme to the PCR tube directly.  (Purification is not necessary at this stage).
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#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
#[[Knight:Electroporation | Transform]] purified DNA into highly competent cells.
#[[Knight:Electroporation | Transform]] purified DNA into highly competent cells.
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#Screen the transformants for the desired mutation using [[Colony PCR  | colony PCR]], [[Restriction Digest | restriction digest]] or [[Sequencing DNA | sequencing]] as appropriate.  Typically 1/4 or 1/8 colonies are correct.
+
#Screen the transformants for the desired mutation using [[Colony PCR  | colony PCR]], [[Restriction Digest | restriction digest]] or [[Sequencing DNA | sequencing]] as appropriate.
==Notes==
==Notes==

Revision as of 15:02, 23 August 2007

Back to Site-directed mutagenesis

Work in progress!

This protocol permits mutation at multiple sites simultaneously with only a single oligo per site.

Contents

Materials

  • Template plasmid purified from a dam+ E. coli strain (not JM110 or SCS110)
  • Mutatagenesis primers (one per mutation, each on the same strand)
  • PfuTurbo DNA polymerase (nonstrand-displacing) and associated reaction buffer
  • Taq ligase
  • dNTPs
  • PCR Thermocycler
  • Dpn I
  • Competent cells

Mutagenesis PCR mix

  • 2.5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
  • X μL (50 ng) of dsDNA template
  • X μL of each oligonucleotide primer
    • For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.
    • If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See Stratagene QuikChange Multi Site-Directed Mutagenesis manual for details.
  • 1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
  • ddH2O to a final volume of 25 μL

Then add

  • 1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
  • 1 μL of Taq Ligase?

Procedure

This procedure is primarily derived from the Stratagene QuikChange Multi Site-Directed Mutagenesis manual with some modifications based on past experience.

  1. Design mutagenesis primers.
    • The primer should be designed so that the desired mutation occurs at the exact center of the primer with 10-15 bp of matching sequence on each side.
    • Primers should be 25-45bp in length with a melting temp of >=75°C. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure. Primers should have comparable melting temperatures.
    • See the Stratagene manual for more detailed information. In particular, adhere to their formula for calculating the melting temperature of your primers and design your primers to have a melting temperature >=75°C.
    • Primers should have at least 40% GC content and terminate in one or more C or G bases at the 3' end.
    • PAGE purification of primers may improve mutagenesis efficiency. See here for links to information on oligo purification.
    • See designing primers for general advice on primer design.
  2. Purify template plasmid from a dam+ E. coli strain via miniprep.
  3. Set up mutagenesis PCR mix as described above.
  4. Run PCR
    1. 95°C for 1 min
    2. 95°C for 1 min
    3. 55°C for 1 min
    4. 65°C for 2 min/kb of plasmid length minimum
    5. Run reaction for 30 cycles.
    • Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
  5. Cool the reaction to <=37°C
  6. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
  7. Incubate 2-3 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
  8. Purify PCR product.
    • I typically do this step using a QIAgen PCR Purification kit but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
  9. Transform purified DNA into highly competent cells.
  10. Screen the transformants for the desired mutation using colony PCR, restriction digest or sequencing as appropriate.

Notes

  • Stratagene does not recommend this protocol for insertions or deletions.
  • Apparently there are no primer spacing-dependent effects on mutagenesis efficiency (primers can be adjacent or far apart).

Reference

QuikChange® Multi Site Directed Mutagenesis Kit

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