Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
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'''Quikchange Site Directed Mutagenesis''' | '''Quikchange Site Directed Mutagenesis''' | ||
* The QuikChange Site-Directed Mutagenesis Kit (Catalog #200519) contains enough reagents for 10 total reactions, | * The QuikChange Site-Directed Mutagenesis Kit (Catalog #200519) contains enough reagents for 10 total reactions, which includes 5 control 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. | ||
* 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 | * Use 125 ng of each primer | ||
* '''Control Reaction''' | * '''Control Reaction''' | ||
5 μl of 10x reaction buffer | ** 5 μl of 10x reaction buffer | ||
2 μl (10 ng) of pWhitescript 4.5-kb control | ** 2 μl (10 ng) of pWhitescript 4.5-kb control template (5 ng/μl) | ||
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 | ** 1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)] | ||
primer #1 [34-mer (100 ng/μl)] | ** 1 μl of dNTP mix | ||
1.25 μl (125 ng) of oligonucleotide control | ** ddH2O to a final volume of 50 μl | ||
primer #2 [34-mer (100 ng/μl)] | |||
1 μl of dNTP mix | |||
ddH2O to a final volume of 50 μl | |||
* '''Sample Reaction''' | * '''Sample Reaction''' | ||
5 μl of 10× reaction buffer | ** 5 μl of 10× reaction buffer | ||
X μl (5–50 ng) of dsDNA template | ** X μl (5–50 ng) of dsDNA template | ||
X μl (125 ng) of oligonucleotide primer #1 | ** X μl (125 ng) of oligonucleotide primer #1 | ||
X μl (125 ng) of oligonucleotide primer #2 | ** X μl (125 ng) of oligonucleotide primer #2 | ||
1 μl of dNTP mix | ** 1 μl of dNTP mix | ||
ddH2O to a final volume of 50 μl | ** 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 | # 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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