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July 24
Started overnight cultures of pSB3K3
July 25
PCR'd parts for pNT002: R0040, K123001, B0014, pSB4A5
PCR'd GFP control insert and pNT003 insert
Gibson assembly and transformation of pNT003 with a negative control
Nanodrop of evaporation concentrated PFGE DNA: 9-1: 11.4ng/uL; 10-2: 29.7ng/uL
Attempt at packaging above DNA with fosmid kit extract, but enzyme wasn't heat inactivated as par procedure
50 mL of phage dillution buffer made for fosmid kit
Evaporation concentrated more PFGE DNA. Nandrop results: 9mix: 14.3ng/uL; 10mix: 2.2ng/uL
Overnight ligation of 9mix and (10mix + 10-2) to redo fosmid packaging, at 16°C
Results
Decided not to use the pNT003 PCR'd insert, as the band of the correct length was much fainter than a lower length band. Chose not to do Gibson assembly of the positive GFP control, despite not having any green colonies last week, instead focusing on assembling pNT002 and pNT003.
 lane 1 NEB 2-log ladder, 2-4 pSB4A5, 5-7 R0040, 8-10 K123001, 11-13 B0014  lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5 R0010, 6 K123003, 7 B0014, 8 blank, 9-11 GFP insert, 12 GFP constitutive promoter, 13 GFP, 14 GFP terminator, 15 NEB 2-log ladder PCR concentrations
| Part |
Concentration (ng/ul) |
| R0040 for pNT002 |
56.4 |
| K123001 |
121.5 |
| B0014 for pNT002 |
100.8 |
| pSB4A5-1 |
98.0 |
| pSB4A5-2 |
97.4 |
July 26
Packaging of 9mix and (10mix + 10-2) ligations
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