July 31
Started a LB+amp overnight culture of the B0034 glycerol stock from the 2010 Caltech iGEM Team
Results
Mass spectrometry did not run correctly; samples did not ionize; must rerun
August 1
PCR pSB3K3 and pNT003 insert with the new primers.
Gel extract the pNT002 insert from the July 29 PCR
Minimal media transfers
Transformation and plating of pUC19 into competent cells for cell dry weight experiment
Overnight culture of B0034 glycerol stock
Overnight ligation of pNT002 insert and PSB3K3 backbone
Restriction digest of parts for pNT002 standard assembly and overnight ligation
Results
No colonies visible on the pUC19-transformed cells
The enrichment culture plates show no visible growth yet.
Gel Extractions of July 29 PCR of pNT002 and pNT003 inserts
| Part |
Concentration (ng/ul) |
| pNT002 insert |
65.9 |
| pNt003 insert |
12.5 |
August 2
Gibson assemble pNT002 using the gel extracted insert and linearized pSB3K3
Gibson assemble pNT003 using the gel extracted insert and linearized pSB4A5
Transform Gibson reactions into our XL-10 cells and Emzo's top ten cells
Replating of more pUC19-transformed competent cells and non-transformed competent cells
Standard Assembly Digest- K12300, B1004, R0010, B0034, along with the destination plasmids.
Run gel of overnight pNT002 ligations
Transformed pNT002 insert+ pSB3K3 ligation
Results
lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 insert with PIPE primers, 8-13 pSB3K3, 14-15 pETDEST53
|
lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 inset with PIPE primers, 8-9 pNT003 insert, 10-11 pNT003 inset with PIPE primers, 12-15 pETDEST53
|
pNT002 ligation showed band on gel
| Part |
Concentration (ng/ul) |
| pSB3k3-1 |
23.8 |
| pSB3K3-2 |
29.9 |
| pETDEST53 |
90.9 |
August 3
Colony PCR of Gibson transforms
Emzo's protocol for traditional assembly of PCR'd pNT002 insert and pSB3K3 plasmid
Results
| Plate |
number of colonies |
| pNT002 + top ten |
55 |
| pNT002 - top ten |
43 |
| pNT002 +XL10 gold |
180 |
| pNT002 - XL10 gold |
400 |
| pNT003 + top ten |
14 |
| pNT003 - top ten |
1 |
| pNT003 +XL10 gold |
0 |
| pNT003 - XL10 gold |
7 |
August 4
Ran gel of colony pcr from yesterday
Results
Some of the colony PCR have insert amplifications of the correct length. Will send some off for sequencing tomorrow.
upper comb lane 1 NEB 2-log ladder, 2-14 pNT002 colonies 1-13 vector amplification; lower comb 1 NEB 2-log ladder, 2-14 pNT002 colonies 1-13 insert amplification
|
lane 1 NEB 2-log ladder, 2-8 pNT002 colonies 14-20 vector amplification, 9-15 pNT002 colonies 14-20 insert amplifications
|
upper comb lane 1 NEB 2-log ladder, 2-13 pNT003 colonies 1-12 vector amplification; lower comb 1 NEB 2-log ladder, 2-13 pNT003 colonies 1-12 insert amplification
|
No growth from traditional assembly on self ligation control or experimental plate
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