User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/09/05: Difference between revisions

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==September 5, 2013==
==September 5, 2013==
* '''QuickChange Site Directed Mutagenesis of V0200 to remove BSMB1 cut site in the Hygro resistance sequence'''
* '''QuickChange Site Directed Mutagenesis of V0200 to remove BSMBI cut site in the Hygro resistance sequence'''
# Use 50 ng of dsDNA template [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/05/06 (BD002)] and 125 ng of each primer.  
# Use 50 ng of dsDNA template [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/05/06 (BD002)] and 125 ng of each primer.  
# Template strands are about 7kb for BD002.
# Template strands are about 7kb for BD002.
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| Total || 50.0  
| Total || 50.0  
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*No control reaction, I will test the accuracy of the mutagenesis with BSMB1 restriction enzyme.
*No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
* Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the sample reaction  
* Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the sample reaction  
* Thermal cycling
* Thermal cycling
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* DpnI Digest (gets rid of methylated template DNA)  
* DpnI Digest (gets rid of methylated template DNA)  
# H2B mut rxn
* Add 1 μL DpnI enzyme to the reaction.
# H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
* Gently and thoroughly mix the reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA  
# LOV mut rxn
 
# LOV control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
'''Transformation'''
* Add 1 μL DpnI enzyme to each sample
* Transform 10μL of Dpn I treated DNA ftom the reaction tube to the 50μL aliquot of XL1-Blue supercompetent cells (Comes with the QuikChange Kit) '''No colony grow'''
* 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  40μL of Dpn I-treated DNA from the reaction tube to the 50μL aliquot of DH5α-T. '''One colony grow'''
* Transform 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells  
*Transform 1μL of control plasmid from the kit to 50μL of XL1-Blue supercompetent cells. '''100s colonies grow'''
*''Do the traditional transformation process: 10minutes on ice, 45 seconds at 42°C, 1 minute on ice, add SOC and incubate 45 minutes at 37°C, Spin down the cells and resuspend the pallet with 100μL LB AMP broth, spread on AMP agar plate and incubate for 18 hours. ''
 


[[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 08:56, 13 October 2013

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September 5, 2013

  • QuickChange Site Directed Mutagenesis of V0200 to remove BSMBI cut site in the Hygro resistance sequence
  1. Use 50 ng of dsDNA template (BD002) and 125 ng of each primer.
  2. Template strands are about 7kb for BD002.
  3. Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μL concentration.
  4. Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μL concentration.
  5. DNA template (BD002) miniprep concentration: 88ng/μL.
Reagents BD002
Plasmid DNA (50 ng) 0.6 μL
primer 1 (10 μM, 125 ng) 2.7
primer 2 (10 μM, 125 ng) 2.66
10x reaction buffer 5.0
dNTP mix 1.0
dH2O 38.04
Total 50.0
  • No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
  • Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the 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)
  • Add 1 μL DpnI enzyme to the reaction.
  • Gently and thoroughly mix the reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA

Transformation

  • Transform 10μL of Dpn I treated DNA ftom the reaction tube to the 50μL aliquot of XL1-Blue supercompetent cells (Comes with the QuikChange Kit) No colony grow
  • Transform 40μL of Dpn I-treated DNA from the reaction tube to the 50μL aliquot of DH5α-T. One colony grow
  • Transform 1μL of control plasmid from the kit to 50μL of XL1-Blue supercompetent cells. 100s colonies grow
  • Do the traditional transformation process: 10minutes on ice, 45 seconds at 42°C, 1 minute on ice, add SOC and incubate 45 minutes at 37°C, Spin down the cells and resuspend the pallet with 100μL LB AMP broth, spread on AMP agar plate and incubate for 18 hours.




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