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July 18
 Colonies 1-6 from Gibson plates; 7 and 8 are reswabs of two colonies. From left to right: DNA ladder,(PNT001; colony 1; vector), P2v, P3v, P4v, P5v, P6v, P7v, P8v, (PNT001; colony 1; insert), P3i, P4i, P5i, P6i  from left to right: DNA ladder, (PNT001; reswab 7; insert), P8i, (16s; colony A; vector), 16Bv, 16Cv, (16s; A; insert), 16Bi, 16Ci
Colony PCR of 16s plasmids, for vector and insert
Gel of short PCR parts to confirm they are being amplified
Pulsed field gel with a larger amount of soil sample DNA
Retransform pSB3K5 into Xl-10s
Results
Gels show mostly expected single bands
July 19
PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control
Results
pSB3K5 plate had 0 colonies.
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.
 lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10 Gibson PCR concentrations
| Part |
Concentration (ng/ul) |
| R0010 for pNT001 |
86.4 |
| B0014 for pNT001 |
128.3 |
| B0014 for pNT003 |
89.1 |
| promoter for GFP Gibson positive control |
51.9 |
| GFP for positive control |
130.4 |
| terminator for positive control |
82.5 |
July 20
Pulse field gel of soil-extracted DNA, in preparation for fosmid insertion
PCR parts for Gibson assembly of pNT001, pNT003 and positive control
Redo Gibson assembly of pNT001 using a smaller total mass of DNA. Gibson assemble GFP from Joe that is known to have worked before as a positive control
Try transforming pSB3K5 with less DNA and as a liquid culture
Results
PCR concentrations
| Part |
Concentration (ng/ul) |
| R0010 for pNT003 |
142.0 |
| K123000 |
182.2 |
| K123003 |
43.7 |
| promoter for GFP Gibson positive control |
74.8 |
| pSB4A5 |
92.8 |
July 21
Gel extraction of 40kb LA River sample DNA from pulse field gel
Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working
Run Gibson assembly of Joe's GFP and pNT001 and stop at different time points. Run on a gel to see what size DNA there is at different times in the reaction
Try "step-wise" Gibson. Add the insert for pNT001 (3 ul) into a 10 ul reaction and incubate at 50˚C for 10 or 20 minutes before adding to a premade Gibson mix of just the vector (2 ul). Continue incubating until a total of 1 hour has passed. This may help with the self-ligation of the vector by giving the insert time to assemble if it works
Results
Gibson Plates
| Plate |
Number of Colonies |
| GFP + |
10 |
| GFP - |
3 |
| pNT001 + |
29 |
| pNT001 - |
2 |
 lane 1 NEB 2-log ladder, 2 GFP 0 min, 3 GFP 5 min, 4 GFP 10 min, 5 GFP 15 min, 6 GFP 30 min, 7 GFP 60 min, 8 blank, 9 pNT001 0 min, 10 pNT001 5 min, 11 pNT001 10 min, 12 pNT001 15 min, 13 pNT001 30 min, 14 pNT001 60 min, 15 NEB 2-log ladder. Triangles represent increasing time (not to scale)
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