Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/25: Difference between revisions
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(Autocreate 2013/04/25 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
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== | ==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, ∞ | |||
* DpnI Digest (gets rid of methylated template DNA) | |||
# H2B mut rxn | |||
# H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR) | |||
# LOV mut rxn | |||
# LOV control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR) | |||
* Add 1 μL DpnI enzyme to each sample | |||
* 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 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells | |||
[[Image:VN_Mutagenesis_Transformation_Trial_1_BL21.png|thumb|frame|center|No colonies were seen the next day.]] | |||
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Revision as of 06:58, 7 May 2013
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April 25, 2013
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