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

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(Autocreate 2013/09/05 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
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=Date=
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==September 5, 2013==
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* '''QuickChange Site Directed Mutagenesis of V0200 to remove BSMB1 cut site in the Hygro resistance sequence'''
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# 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.
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# Template strands are about 7kb for BD002.
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#Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μL concentration.
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#Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μL concentration.
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#DNA template (BD002) miniprep concentration: 88ng/μL.
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'''List title'''
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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# List items
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|-
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| Reagents || BD002
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|-
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| Plasmid DNA (50 ng) || 0.6 μL
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|-
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| primer 1 (10 μM, 125 ng) || 2.7
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|-
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| primer 2 (10 μM, 125 ng) || 2.66
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|-
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| 10x reaction buffer|| 5.0
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|-
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| dNTP mix || 1.0
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|-
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| dH<sub>2</sub>O || 38.04
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|-
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| Total || 50.0
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|}
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*No control reaction, I will test the accuracy of the mutagenesis with BSMB1 restriction enzyme.
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* Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the sample reaction
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* Thermal cycling
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** 95°C/ 30 sec
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** [95°C/ 30 sec; 55°C/ 1 min; 68°C/ '''7''' min (1 min/kb plasmid length)]x18
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** 4°C, ∞
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* DpnI Digest (gets rid of methylated template DNA)
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# H2B mut rxn
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# H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
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# LOV mut rxn
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# LOV control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
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* Add 1 μL DpnI enzyme to each sample
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* 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
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* Transform 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells
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[[Image:VN_Mutagenesis_Transformation_Trial_1_BL21.png‎|thumb|frame|center|No colonies were seen the next day.]]
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|}
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__NOTOC__

Revision as of 18:13, 5 September 2013

PcTF Subcloning in E. coli Main project page
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September 5, 2013

  • QuickChange Site Directed Mutagenesis of V0200 to remove BSMB1 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 BSMB1 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)
  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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