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)
 
(2 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"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#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 19, 2013==
==April 22, 2013==
'''Quikchange Site Directed Mutagenesis'''
'''Quikchange Site Directed Mutagenesis'''


Line 12: Line 12:
* 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
* '''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'''
* '''Control Reaction'''

Latest revision as of 22:39, 26 September 2017

Project name Main project page
Previous entry      Next entry

April 22, 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
  • 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
  1. Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
  2. Thermal cycling: 95°C, 30 sec; [95°C, 30 sec; 55°C, 1 min; 68°C, 7 min (1 min/kb plasmid length)]x18
  3. Add 1 μl of the Dpn I restriction enzyme (10 U/μl)
  4. 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
  5. 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
  6. 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
  7. Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes
  8. Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes
  9. 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
  10. Plate entire 250 μL volume and incubate for at least 16 hours


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes/2013/04/22&oldid=1012480"