Biofilm Track
Melody
Biofilm Protocol Day 1
- Note: Each "batch" of O/N cultures include one test tube of control, RN4220, and 8325-4
- Note: I used the timer/clock on the lab bench for the timing
- Batch 1: in @ 4:03 p.m.
- Batch 2: in @ 5:10 p.m.
- Batch 3: in @ 6:06 p.m.
- Batch 4: in @ 4:03 p.m.
- Batch 5: in @ 4:03 p.m.
- Batch 6: in @ 4:03 p.m.
dspB Track
- 1/100 dilution of pre-dspB and Pcon in 37C shaking incubator at 12:10pm
- O/N culture in 4C
- 1/100 dilution of Pcon growing in 37C shaking (AMP not CHLOR) at 3:30pm
- 1/100 dilution of:
- pre-dspB OD600=1.054
- P-con OD600=1.344
PCR using Rafael's method Attempt #5
- Using new PNKed primers and Taq
PCR Master mix
| Reagent | 1x rxn volume (uL) |
| 10x Thermopol buffer | 2.5 |
| 10mM dNTP | 0.5 |
| sdH2O | 13.65 |
| Taq Polymerase | 0.1 |
| MgCl2 | 2 |
| DMSO - 5% | 1.25 |
| gDNA | 1 |
| fw rxn 2 | 2 |
| re rxn 2 | 2 |
| Total | 25 | PCR Cycles:
- 95C @ 2min
- Cycle 30x:
- 95C @ 30 sec
- 58.5C @ 10 sec
- 72C @ 80 sec
- 72C @ 10 min
- 10C @ hold
Start: 4:17pm
End: 5:48pm
Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Machine conditions: 0.5x TBE buffer, 100V, 45min
Gel orientation:
Gel orientation
| A | B | C | D | E | 100bp ladder | F | G | H | I | W | 1 kb ladder | Results:
cPCR NCC colonies to verify before making glycerol stock
PCR Master mix
| Reagent | 1x rxn volume (uL) |
| 5x Reaction buffer | 5 |
| 10mM dNTP | 0.5 |
| sdH2O | 12.15 |
| Phusion Pol | 0.1 |
| MgCl2 | 2 |
| DMSO - 5% | 1.25 |
| G1004 | 2 |
| G1005 | 2 |
| Total | 25 | - Tubes: NCC, Poco, Pcon, W
- Poco and Pcon are for QS track
PCR Cycles:
- 98C @ 3min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 7:26pm
End: 8:29pm
Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Machine conditions: 0.5x TBE buffer, 100V, 60min
Gel orientation:
Gel orientation
| NCC | Poco | 100bp ladder | Pcon | W | Results:
O/N culture
- O/N culture of NCC made with 1.5mL LB broth for glycerol stock
- used 1.5uL of C1 antibiotic
- put into 37C shaking incubator at 6:40pm
|
iGEM Project name 1
