Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
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* The QuikChange Site-Directed Mutagenesis Kit (Catalog #200519) contains enough reagents for 10 total reactions, which includes 5 control reactions. | * 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 | * 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. | ||
to multiple freeze-thaw cycles. | |||
* Use 125 ng of each primer | * Use 125 ng of each primer | ||
* '''Control Reaction''' | * '''Control Reaction''' | ||
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# Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction | # 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 | # 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) | # Add 1 μl of the Dpn I restriction enzyme (10 U/μl) |
Revision as of 07:19, 22 April 2013
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April 19, 2013Quikchange Site Directed Mutagenesis
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