Difference between revisions of "User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/13"

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(Autocreate 2013/11/13 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
 
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'''List title'''
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'''Troubleshooting BD002 PCR Linearization for GG Assembly'''
# List items
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Date'''
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| align="center" style="background:#f0f0f0;"|'''Plasmid Conc.'''
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| align="center" style="background:#f0f0f0;"|'''Therm Cycling Conditions'''
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| align="center" style="background:#f0f0f0;"|'''Gel Picture'''
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| align="center" style="background:#f0f0f0;"|'''Comment'''
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|-
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| 11/9/2013||9 ng Plasmid: BD002||[95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]||||
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|-
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| 11/9/2013||9 ng Plasmid: KAH187 (CMV)||[95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]||||
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|-
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| 11/10/2013||0.09ng Plasmid: BD002||[95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]||||Gel Extract the band close to 5K bps No result
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|-
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| 11/10/2013||0.09ng Plasmid: BD002||[95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]||||Amplified with V0200 Sequencing Primers to make sure Kozak-HSV-CFP-NLS-Stop-PolyA (2K bps are there).
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|-
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| 11/11/2013||Plasmid: BD002||[95C (30 sec.), 49C (45 Sec.), 72C (6 min.)]||||Column 1,2: 9 ng Plasmid, Column 3,4: 0.09 ng Plasmid, Column 5,6: 0.5 ml primer from 10mM solution 
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|-
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| 11/11/2013||0.09ng Plasmid: BD002||[95C (30 sec.), 49C (45 Sec.), 75C (6 min.)]||||Increase the extention temperature to 75C and annealing temperature to 49C
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|-
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| 11/11/2013||0.09ng Plasmid: BD002||[95C (30 sec.), 55C (45 Sec.), 72C (6 min.)]||||Increase the annealing temperature to 55C
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|-
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|}

Revision as of 11:36, 13 November 2013

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Date

Troubleshooting BD002 PCR Linearization for GG Assembly


Date Plasmid Conc. Therm Cycling Conditions Gel Picture Comment
11/9/2013 9 ng Plasmid: BD002 [95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]
11/9/2013 9 ng Plasmid: KAH187 (CMV) [95C (30 sec.), 45C (45 Sec.), 72C (6 min.)]
11/10/2013 0.09ng Plasmid: BD002 [95C (30 sec.), 45C (45 Sec.), 72C (6 min.)] Gel Extract the band close to 5K bps No result
11/10/2013 0.09ng Plasmid: BD002 [95C (30 sec.), 45C (45 Sec.), 72C (6 min.)] Amplified with V0200 Sequencing Primers to make sure Kozak-HSV-CFP-NLS-Stop-PolyA (2K bps are there).
11/11/2013 Plasmid: BD002 [95C (30 sec.), 49C (45 Sec.), 72C (6 min.)] Column 1,2: 9 ng Plasmid, Column 3,4: 0.09 ng Plasmid, Column 5,6: 0.5 ml primer from 10mM solution
11/11/2013 0.09ng Plasmid: BD002 [95C (30 sec.), 49C (45 Sec.), 75C (6 min.)] Increase the extention temperature to 75C and annealing temperature to 49C
11/11/2013 0.09ng Plasmid: BD002 [95C (30 sec.), 55C (45 Sec.), 72C (6 min.)] Increase the annealing temperature to 55C