Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
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to multiple freeze-thaw cycles. | to multiple freeze-thaw cycles. | ||
* Use 125 ng of each primer | * Use 125 ng of each primer | ||
* 10× Reaction Buffer | * '''10× Reaction Buffer''' | ||
** 100 mM KCl | ** 100 mM KCl | ||
** 100 mM(NH4)2SO4 | ** 100 mM(NH4)2SO4 | ||
Line 20: | Line 20: | ||
** 20 mM MgSO4 | ** 20 mM MgSO4 | ||
** 1% Triton® X-100 | ** 1% Triton® X-100 | ||
** 1 mg/ml nuclease-free bovine serum albumin | ** 1 mg/ml nuclease-free bovine serum albumin (BSA) | ||
(BSA) | |||
* TE Buffer | * '''TE Buffer''' | ||
** 10 mM Tris-HCl (pH 7.5) | ** 10 mM Tris-HCl (pH 7.5) | ||
** 1 mM EDTA | ** 1 mM EDTA | ||
* '''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 |
Revision as of 06:55, 22 April 2013
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April 19, 2013Quikchange Site Directed Mutagenesis
which includes 5 control reactions.
to multiple freeze-thaw cycles.
|