User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/28

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(Autocreate 2013/10/28 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
Current revision (15:39, 30 October 2013) (view source)
(Colony PCR of Golden Gate Assembly of BD003 and BD004)
 
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=Date=
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=Colony PCR of [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/25 Golden Gate Assembly] of BD003 and BD004=
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'''List title'''
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* Pick 10 colonies of each plate and grow in 2 mL LB AMP broth for 6 hours.
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# List items
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* Spin down the cells for 3 minutes in microcentrifuge with top speed.
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* Resuspend the pallet in 100μL dH2O.
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* Incubate the cells in 98°C for 5 minutes.
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* Spin down the cells for 3 minutes in microcentrifuge with top speed.
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* Take the supernatent as DNA template for PCR reactions.
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* PCR colonies at: 95°C for 3 min. , [95°C 3 min, 45°C 45 sec., 72°C 3 min.] ×35, 72°C 6min. 4°C ∞.
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{| {{table}} ; style="text-align:center; width:550px; height:200px;"
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|[[Image:20131029 103655 (3).jpg|350px|BD004(HPK promoter) bands shows that only the promoter part is amplified (588bps)]] ||[[Image:20131029 103655 (4).jpg |350px|salam]]
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|-
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|BD004('''HPK''' promoter) bands shows that only the promoter part is amplified (588bps) || BD003('''CMV''' promoter) bands shows that only the promoter part is amplified (~ 600bps)
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|}

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Colony PCR of Golden Gate Assembly of BD003 and BD004

  • Pick 10 colonies of each plate and grow in 2 mL LB AMP broth for 6 hours.
  • Spin down the cells for 3 minutes in microcentrifuge with top speed.
  • Resuspend the pallet in 100μL dH2O.
  • Incubate the cells in 98°C for 5 minutes.
  • Spin down the cells for 3 minutes in microcentrifuge with top speed.
  • Take the supernatent as DNA template for PCR reactions.
  • PCR colonies at: 95°C for 3 min. , [95°C 3 min, 45°C 45 sec., 72°C 3 min.] ×35, 72°C 6min. 4°C ∞.


BD004(HPK promoter) bands shows that only the promoter part is amplified (588bps) salam
BD004(HPK promoter) bands shows that only the promoter part is amplified (588bps) BD003(CMV promoter) bands shows that only the promoter part is amplified (~ 600bps)
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