December 5, 2012
11/21/12
Results from last week's 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
- ---Karmella 15:56, 21 November 2012 (EST): Digest, purification, and ligation
Assemblies
- NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
- NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
- NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
- Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
- Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
- Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
- Digests:
- In a 30 μL reaction, cut H2B with XbaI and SpeI
- In a 30 μL reaction, cut LOV with XbaI and SpeI
| Reagent |
H2B PCR |
LOV PCR
|
| DNA |
15.0 |
15.0
|
| XbaI |
1.0 |
1.0
|
| SpeI |
1.0 |
1.0
|
| 10x buffer |
3.0 |
3.0
|
| dH2O |
10.0 |
10.0
|
| Total vol. |
30. 0 |
30.0
|
- Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
- Cut band out of gel (use UV box)
Results from gel recovery:
| Sample |
260 |
260/280 |
ng/μL
|
| H2B - X/S 1 |
0.016 |
1.807 |
15.961
|
| H2B - X/S 2 |
0.009 |
1.976 |
8.619
|
| LOV - X/S 1 |
0.006 |
1.694 |
6.455
|
| LOV - X/S 2 |
0.025 |
1.883 |
25.363
|
- H2B = 410
- LOV = 461
- There were two samples made of each PCR reaction. The two from the H2B are shown on the left, and the two from the LOV are shown on the right.
Dephosphorylation (11/23/12)
> Dephosphorylation (Roche)
- pSB1A3 - X/S = 2000 bp
| Reagent |
Volume
|
| DNA (clean digest) |
11.0 (~200 ng)
|
| 10x phos. buffer |
1.5
|
| phosphatase |
0.5
|
| dH2O |
2.0
|
| |
15 μL
|
- Incubate at 37°C/ 10 min.
- Heat inactivate at 75°C/ 2 min.
- [final] = 200 ng/μL / 15 μL = ~13 ng/μL
Ligation
- Use 20 ng of vector for each ligation = 1.5 μL pSB1A3
- Use 3x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least 0.8 μL H2B
- Use 3x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least 0.5 μL LOV
| |
H2B |
LOV |
Negative Control |
H2B |
LOV |
Negative Control
|
| Insert DNA (2x mol vector) |
1.0 μL |
1.0 |
--- |
1.0 |
1.0 |
---
|
| Vector DNA (50 ng) |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5
|
| 2x Roche Rapid Ligation buffer |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase |
1.0 NEB |
1.0 NEB |
1.0 NEB |
1.0 Roche |
1.0 Roche |
1.0 Roche
|
| dH2O |
1.5 |
1.5 |
2.5 |
1.5 |
1.5 |
2.5
|
| TOTAL |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0
|
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.
|
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
|
Project name
