User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/13

PcTF Subcloning in E. coli

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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