11/21/12
Results from yesterday's ligation
- Got 1:1 ratio of ligation: negative control for both
- About 10 colonies total on every plate
- Forgot to take into account X/S vector self-ligation
- Will pick 4 colonies from each (H2B, LOV) to see if anything worked.
- In the mean time, will retry cloning, trouble shoot procedure with help from Dr. Haynes
- Repeat PCR of H2B and LOV inserts
- Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
- H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
- LOV plasmid + BB_LOV fwd + BB_LOV rev
| 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
- H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
- LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
- (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 trials 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
> Dephosphorylation (Roche)
- pSB1A3 - X/S =
| Reagent |
Volume
|
| DNA (clean digest) |
up to 11.0 (~200 ng)
|
| 10x buffer d.p. |
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 2x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL H2B = ### μL H2B
- Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL LOV = ### μL LOV
| |
H2B |
LOV |
Negative Control
|
| Insert DNA (2x mol vector) |
### μL |
### |
---
|
| Vector DNA (50 ng) |
1.5 |
1.5 |
1.5
|
| 2x Roche Rapid Ligation buffer |
5.0 |
5.0 |
5.0
|
| New England Biolabs T4 ligase |
1.0 |
1.0 |
1.0
|
| dH2O |
### |
### |
###
|
| TOTAL |
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
|
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