02/23/13
- Type IIS Assembly, PCR: H2B-LOV+his
Type IIS Assembly, PCR: LOV-H2B+his
- H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
- LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag and TAA stop (21 bp)
- pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
- Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.
PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Reagent
|
Volume
|
Expected: 1. H2B = 378 2. LOV = 450 3, 4. pSB1A3 = ~2000
|
10 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
0.2 μL
|
primer 1 |
1.0
|
primer 2 |
1.0
|
2x GoTaq |
25.0
|
dH2O |
22.8
|
|
50.0 μL
|
Purification
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA]
Sample
|
OD 260
|
260/280
|
ng/μL
|
1. H2B |
0.04 |
1.667 |
40.4
|
2. LOV+his |
0.019 |
1.562 |
18.6
|
3. pSB1A3 |
0.069 |
1.754 |
69.4
|
4. pSB1A3 |
0.085 |
1.817 |
85.5
|
Dilutions: to final concentration of 20 fmol/μL (40 μL)
- x = length in bp ÷ measured ng/μL * 0.013 * 40
DNA
|
length (bp)
|
ng/μL
|
Vol. (x)
|
+ dH2O
|
pSB1A3 |
2000 |
85.5 |
12.2 |
27.8
|
H2B |
378 |
40.4 |
4.9 |
35.1
|
LOV+his |
450 |
18.6 |
12.6 |
27.4
|
(02/25/13)
Golden Gate Reactions
- H2B + LOV+his + pSB1A3
- pSB1A3
- H2B + LOV+his + pSB1A3, 2xDNA
- pSB1A3, 2xDNA
Reagent |
1 |
2 |
Master mix (x3) |
3 |
4 |
Master mix (x3)
|
pSB1A3 gg |
1.0 |
1.0 |
--- |
2.0 |
2.0 |
---
|
H2B gg |
1.0 |
H2O |
--- |
2.0 |
H2O |
---
|
LOV+his gg |
1.0 |
H2O |
--- |
2.0 |
H2O |
---
|
10x PRO ligase buffer |
1.0 |
1.0 |
3.0 |
1.0 |
1.0 |
3.0
|
NEB T4 lgase |
0.25 |
0.25 |
0.75 |
0.25 |
0.25 |
0.75
|
BsmBI |
0.5 |
0.5 |
1.5 |
0.5 |
0.5 |
1.5
|
dH2O |
5.25 |
5.25 |
15.75 |
2.25 |
2.25 |
6.75
|
|
10.0 |
10.0 |
|
10.0 |
10.0 |
|
- For 1 and 2, aliquot 7.0 mix to each tube; add DNA/ H2O
- For 3 and 4, aliquot 4.0 mix to each tube; add DNA/ H2O
Thermal cycling
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
|