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 | |||
* 10× Reaction Buffer | |||
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) | |||
* TE Buffer | |||
10 mM Tris-HCl (pH 7.5) | |||
1 mM EDTA | |||
# '''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 06:45, 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
plasmid (0.1 ng/μl) to a 50-μl aliquot of the supercompetent cells
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