Biofilm Track
Melody
- Took O/N cultures out of shaking and non-shaking incubator @ 10:00 a.m.
- No contamination in both controls
- Took out plate 100830M120 @ 10:00 a.m.
- Took out plate 100830M222 @ 12:00 p.m.
- contamination in D7 (characteristic white colonies)
Biofilm Protocol Day 3
- 3 x 300 ul PBS wash on plates 100830M120 + 100830M222
- 100830M120 heat fixed @ 11:55 a.m.
- 100830M222 heat fixed @ 12:50 p.m.
Biofilm Protocol Day 2
- Innoculate into plates 100831M24 + 100831M26
100831M24 + 100831M26 Plate Layout
| Row |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
| C |
R |
R |
C |
R |
C |
8 |
8 |
C |
| D |
R |
C |
R |
R |
8 |
C |
8 |
8 |
| E |
R |
R |
C |
R |
8 |
8 |
8 |
8 |
- 100831M26 incubated @ 2:30 p.m.
- 100831M24 incubated @ 3:20 p.m.
Biofilm Day 4 continued
- Initial OD550 readings on 100824E + 100824M
100824E Data
| 25 |
<> |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|---|
| 26 |
C |
0.088 |
0.1711 |
0.0871 |
0.1781 |
0.26520 |
0.0876 |
0.201 |
0.0899 |
|---|
| 27 |
D |
0.1575 |
0.0877 |
0.159 |
0.178 |
0.0885 |
0.2086 |
0.2007 |
0.2098 |
|---|
| 28 |
E |
0.1404 |
0.1722 |
0.0877 |
0.09 |
0.0911 |
0.1961 |
0.0878 |
0.1733 |
|---|
100824M Data
| 25 |
<> |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|---|
| 26 |
C |
0.1 |
0.2297 |
0.1008 |
0.1596 |
0.2858 |
0.107 |
0.30880 |
0.1014 |
|---|
| 27 |
D |
0.1701 |
0.1034 |
0.2243 |
0.2292 |
0.1074 |
0.2834 |
0.2811 |
0.2646 |
|---|
| 28 |
E |
0.2613 |
0.28 |
0.129 |
0.1379 |
0.1135 |
0.32410 |
0.1013 |
0.2958 |
|---|
- Resolubilized in 95% ethanol
100824ERS
</tr
| 25 |
<> |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|---|
| 26 |
C |
0.0858 |
0.29070 |
0.0824 |
0.2807 |
0.31060 |
0.0869 |
0.2732 |
0.0899 |
|---|
| 27 |
D |
0.2961 |
0.0886 |
0.2548 |
0.2782 |
0.0802 |
0.32320 |
0.36630 |
0.2829 |
|---|
| 28 |
E |
0.25890 |
0.3078 |
0.1923 |
0.2443 |
0.0957 |
0.333 |
0.0831 |
0.3118 |
|---|
100824MRS
| 25 |
<> |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|---|
| 26 |
C |
0.104 |
0.2175 |
0.1014 |
0.2176 |
0.2177 |
0.1105 |
0.1784 |
0.1171 |
|---|
| 27 |
D |
0.1705 |
0.0967 |
0.1966 |
0.2031 |
0.1184 |
0.2355 |
0.2417 |
0.2489 |
|---|
| 28 |
E |
0.2067 |
0.226 |
0.1062 |
0.1086 |
0.0997 |
0.2381 |
0.1079 |
0.1886 |
|---|
Biofilm Protocol Day 1
- O/N cultures of RN4220 and 8325-4 incubated @ 4:40 p.m. w/o shaking
TSA plates
- TSA plate spread with RN4220 and 8325-4
Autoclaved Tips
- 300ul, 1000ul, 10ul pipette tips autoclaved
dspB Track
Restriction Digest
Restriction Digest Supermix
| REAGENTS | 1 RXN VOLUME (uL) |
| Buffer 2 | 5 | |
| BSA | 0.5 |
- DNA, add...
- His: 9.5uL
- No his: 9.5uL
- RFP chlor bb: 7uL
- ccdB chlor bb: 7uL
- Enzymes: EecoRI and SpeI (1uL each)
- Total volume: RFP chlor backbone (bb): 14.50 + ccd chlor bb: 14.50
- Add 30uL H2O to each digest after 1 hour
Gel verification on RD
- Protocol: biobrick "digest" protocol
- Changes: load 15uL into each well
Gel orientation:
Gel orientation
| ccdb chlorbb | ccdb chlorbb | ccdb chlorbb | 100bp ladder | RFP chlorbb | RFP chlorbb | RFP chlorbb | 100bp ladder | EP chlor | EP chlor | EP chlor | Machine conditions: 0.5x TBE buffer, 100V, 60min
- RFP chlor: 0.4g
- ccdb chlor: 0.5g
- ccdb E/P: 0.8g
Ligations
- Protocol: biobrick method
- His + ccdB -> HC
- His + RFP -> HR
- no his + ccdb -> NHC
- no his + RFP -> NHR
Calculations
[math]\displaystyle{ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng) }[/math]
[math]\displaystyle{ 3 \times \frac{1200}{2400} \times = ng }[/math]
[math]\displaystyle{ 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }[/math]
- Where x = concentration of insert
- gel extraction failed. No ligations conducted.
|
iGEM Project name 1
