dspB Track
Plate results:
- 50uL HR - no colonies
- 450uL HR - ~20 colonies (most of them red); (A, D - white?; B, C - pink?)
- 50uL HC - no colonies
- 450uL NC - no colonies
- 50uL NR - 3 colonies (2red) (A)
- 450uL NR - ~20 colonies (~ half of them red) (A, B, C, D)
- 50uL NC - colonies
- 450uL NC - 34 colonies (A, B, C, D)
Colony PCR
PCR Master mix
Reagent | 1x rxn volume (uL) | Master Mix |
5x rxn buffer | 5 | x16 | 80 |
10mM dNTP | 0.5 | x16 | 8 |
sdH2O | 12.15 | x16 | 194.4 |
Phusion polymerase | 0.1 | x16 | 1.6 |
MgCl2 | 2 | x16 | 32 |
DMSO - 5% | 1.25 | x16 | 20 |
10uM fw primer (G1004) | 2 | x16 | 32 |
10uM re primer (G1005) | 2 | x16 | 32 |
Total | 25 | | 400 |
- Pick colony from respective plate and place in respective tubes
PCR Cycles:
- 98C @ 3min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 1:53pm
End: 2:57pm
Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Changes: 0.6g agarose/70mL 0.5x TBE buffer
- Machine conditions: 0.5x TBE buffer, 100V, 50min
Gel orientation:
Gel orientation
450uL NR A | 450uL NR B | 450uL NR C | 450uL NR D | 50uL NR A | 450uL NC A | 450uL NC B | 100 bp ladder | 450uL NC C | 450uL NC D | 450uL HR A | 450uL HR B | 450uL HR C | 450uL HR D | W |
Results:
Re-Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Changes: 0.6g agarose/70mL 0.5x TBE buffer
- Machine conditions: 0.5x TBE buffer, 100V, 50min
Gel orientation:
Gel orientation
450uL NC A | 450uL NC B | 450uL NC C | 450uL NC D | 50uL HR A | 450uL HR B | 450uL HR C | 100 bp ladder | 450uL HR D C | 450uL NR A | 450uL NR B | 450uL NR C | 450uL NR D | 50uL NR A | W | 1kb ladder |
Results:
Began O/N Culture
- in 37C shaking incubator at 11:30pm
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