December 5, 2012
Results from last 11/23/12 ligation
- None of the colonies on any plates grew
- Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
- Repeat PCR of H2B and LOV inserts with new primers
- Note: Primers add a XbaI site upstream and the SpeI site downstream
- Final Gibson assembly products
- pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
- pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
- H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
- LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
- H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
- LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev
Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct
| Reagents |
H2B |
LOV
|
| Plasmid DNA |
0.5 μL |
0.5
|
| primer 1 (10 μM) |
1.0 |
1.0
|
| primer 2 (10 μM) |
1.0 |
1.0
|
| 2x GoTaq mix |
12.5 |
12.5
|
| dH2O |
10.0 |
10.0
|
| Total |
25.0 |
25.0
|
PCR program:
- 95°C, 3 min.
- [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
- 72°C, 3 min.
- 4°C ∞
- Clean up PCR products using the Zymo Clean and Concentrator kit
- Be sure to elute using 15 μL dH2O
| Construct |
Product |
260 |
280 |
260/280 |
ng/μL
|
| H2B-LOV |
H2B 1 |
0.061 |
0.033 |
1.863 |
60.545
|
| H2B-LOV |
H2B 2 |
0.056 |
0.03 |
1.857 |
56.22
|
| H2B-LOV |
LOV 1 |
0.063 |
0.033 |
1.903 |
62.673
|
| H2B-LOV |
LOV 2 |
0.06 |
0.04 |
1.85 |
59.844
|
| LOV-H2B |
H2B 1 |
0.072 |
0.033 |
1.807 |
72.381
|
| LOV-H2B |
H2B 2 |
0.06 |
0.036 |
1.799 |
59.925
|
| LOV-H2B |
LOV 1 |
0.006 |
0.036 |
1.849 |
65.881
|
| LOV-H2B |
LOV 2 |
0.065 |
0.035 |
1.839 |
65.285
|
|
Project name
