Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/22: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav) |
|||
(6 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| 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''' | * '''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) |
Latest revision as of 22:39, 26 September 2017
Project name | Main project page Previous entry Next entry |
April 22, 2013Quikchange Site Directed Mutagenesis
|