Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/25

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Current revision (08:58, 7 May 2013) (view source)
(April 25, 2013)
 
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==April 25, 2013==
==April 25, 2013==
* '''QuickChange Site Directed Mutagenesis'''
* '''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.
+
* Use 50 ng of dsDNA template and 125 ng of each primer.  
 +
* Template strands are about 5kb for H2B-GFP and 7kb for LOV.
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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* 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
* Thermal cycling
-
** 95°C/30 sec
+
** 95°C/ 30 sec
-
** [95°C/30 sec; 55°C/1 min; 68°C/7 min (1 min/kb plasmid length)]x18  
+
** [95°C/ 30 sec; 55°C/ 1 min; 68°C/ 7 min (1 min/kb plasmid length)]x18  
** 4°C, ∞
** 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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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.
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
dH2O 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)
  1. H2B mut rxn
  2. H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
  3. LOV mut rxn
  4. 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
No colonies were seen the next day.
No colonies were seen the next day.


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