Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
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== | ==April 19, 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 | |||
# Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction | |||
# Thermal cycling: 95°C, 30 sec; [95°C, 30 sec; 55°C, 1 min; 68°C, 7 min (1 min/kb plasmid length)]x18 | |||
# Add 1 μl of the Dpn I restriction enzyme (10 U/μl) | |||
# 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 | |||
# 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 | |||
# 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 | |||
# Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes | |||
# Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes | |||
# 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 | |||
# Plate entire 250 μL volume and incubate for at least 16 hours | |||
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Revision as of 07:19, 22 April 2013
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April 19, 2013Quikchange Site Directed Mutagenesis
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