Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(11 intermediate revisions by the same user not shown) | |||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==April | ==April 22, 2013== | ||
'''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''' | |||
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''' | |||
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) | ||
# 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 06:47, 7 May 2013
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
April 22, 2013Quikchange Site Directed Mutagenesis
|