Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/25: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2013/04/25 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
|||
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 25, 2013== | ||
* | * '''QuickChange Site Directed Mutagenesis''' | ||
* Use 50 ng of dsDNA template and 125 ng of each primer. Template strands are about 5kb for H2B-GFP and 7kb for LOV. | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Reagents || H2B || LOV | |||
|- | |||
| Plasmid DNA (50 ng) || 0.4 μL || 0.2 | |||
|- | |||
| primer 1 (10 μM, 125 ng) || 1.7 || 1.7 | |||
|- | |||
| primer 2 (10 μM, 125 ng) || 1.7 || 1.7 | |||
|- | |||
| 10x reaction buffer|| 5.0 || 5.0 | |||
|- | |||
| dNTP mix || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 40.2 || 40.4 | |||
|- | |||
| Total || 50.0 || 50.0 | |||
|} | |||
* 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 | |||
** 4°C, ∞ | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 13:07, 25 April 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 25, 2013
|