Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
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1 mM EDTA | 1 mM EDTA | ||
* '''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 | ||
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ddH2O to a final volume of 50 μl | ddH2O to a final volume of 50 μl | ||
* '''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 | ||
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# 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 | # 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 | # 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 | # 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 | ||
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 | # 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 | # Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes |
Revision as of 06:46, 22 April 2013
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April 19, 2013Quikchange Site Directed Mutagenesis
which includes 5 control reactions.
to multiple freeze-thaw cycles.
100 mM KCl 100 mM(NH4)2SO4 200 mM Tris-HCl (pH 8.8) 20 mM MgSO4 1% Triton® X-100 1 mg/ml nuclease-free bovine serum albumin (BSA)
10 mM Tris-HCl (pH 7.5) 1 mM EDTA
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
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
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