Biofilm Track
Picked two colonies (1 from each TSA plates preped by Jason and Melody) and placed in tube with 5ml TSB.
for ON culture growth. (static condition, 37C, 20hours)
DspB Track
Marianne, Vicki
PCR gDNA (HK1561)
- Resuspend primers in water to a concentration of 100uM
- Primers:
- Upstream dspB His: 0.285mL water + 28.5nmol primer
- Upstream dspB: 0.312mL water + 31.2nmol primer
- Downstream dspB: 0.411mL water + 41.1nmol primer
- DspB fw rxn1: 0.290mL water + 29.0nmol primer
- DspB rev rxn1: 0.387mL water + 38.7nmol primer
- DspB fw rxn2: 0.342mL water + 34.2nmol primer
- DspB rev rxn2: 0.431mL water + 43.1nmol primer
- Rehydrate Aggregatibacter actinomycetemcomitans: 250uL sdH2O into vial with 5ug of dried DNA. Vial in 37C for 1 hour.
Protocol: Common protocols for PCR
PCR Master mix
| Reagent | 1x rxn volume (uL) | Master Mix |
| 5x rxn buffer | 5 | x15 | 75 |
| 10mM dNTP | 2 | x15 | 30 |
| sdH2O | 8.8 | x15 | 132 |
| Phusion polymerase | 0.2 | x15 | 3 |
| DNA | 5 | x15 | 75 |
| Total | 21 | | 315 | - Add 2uL of fw and rev primers to each PCR tube.
- Elongation time: 40s
PCR Tubes
| Label | Content |
| A | dspB His 1 |
| B | dspB His 2 |
| C | dspB His 3 |
| D | dspB no His 1 |
| E | dspB no His 2 |
| F | dspB no His 3 |
| G | dspB rxn1 1 |
| H | dspB rxn1 2 |
| I | dspB rxn1 3 |
| J | dspB rxn2 1 |
| K | dspB rxn2 2 |
| L | dspB rxn2 3 |
| M | H2O control |
PCR Cycles:
- 98C @ 30s
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 1422
End: 1522
Vicki Ma 03:00, 3 August 2010 (EDT)
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iGEM Project name 1
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