Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions

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==April 19, 2013==
==April 22, 2013==
'''Quikchange Site Directed Mutagenesis'''
'''Quikchange Site Directed Mutagenesis'''


Revision as of 06:47, 7 May 2013

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April 22, 2013

Quikchange Site Directed Mutagenesis

  • The QuikChange Site-Directed Mutagenesis Kit (Catalog #200519) contains enough reagents for 10 total reactions, which includes 5 control reactions.
  • Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mix to multiple freeze-thaw cycles.
  • Use 125 ng of each primer
  • Control Reaction
    • 5 μl of 10x reaction buffer
    • 2 μl (10 ng) of pWhitescript 4.5-kb control template (5 ng/μl)
    • 1.25 μl (125 ng) of oligonucleotide control primer #1 [34-mer (100 ng/μl)]
    • 1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)]
    • 1 μl of dNTP mix
    • ddH2O to a final volume of 50 μl
  • Sample Reaction
    • 5 μl of 10× reaction buffer
    • X μl (5–50 ng) of dsDNA template
    • X μl (125 ng) of oligonucleotide primer #1
    • X μl (125 ng) of oligonucleotide primer #2
    • 1 μl of dNTP mix
    • ddH2O to a final volume of 50 μl
  1. Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
  2. Thermal cycling: 95°C, 30 sec; [95°C, 30 sec; 55°C, 1 min; 68°C, 7 min (1 min/kb plasmid length)]x18
  3. Add 1 μl of the Dpn I restriction enzyme (10 U/μl)
  4. Gently and thoroughly mix each reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA
  5. Transform 1 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of XL1-Blue supercompetent cells
  6. As an optional control, verify the transformation efficiency of the XL1-Blue supercompetent cells by adding 1 μl of the pUC18 control plasmid (0.1 ng/μl) to a 50-μl aliquot of the supercompetent cells
  7. Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes
  8. Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes
  9. Add 0.5 ml of NZY+ broth preheated to 42°C and incubate the transformation reactions at 37°C for 1 hour with shaking at 225–250 rpm
  10. Plate entire 250 μL volume and incubate for at least 16 hours


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