08/07/11
- ✓ Cultures/minipreps: KAH235(2), retransformations DPRE, MV1
- ✓ Assemblies: PCR inserts for pSB31 cloning
Minipreps
> Check 1-3 with E/S digests (DPRE has PstI sites)
> Check 4 w/ E/P
| Reagent |
Volume
|
Expected:
1,2. KAH235 (E/S) = 2787
3. DPRE (E/P) = 1740
4. MV1 (E/S) = 4102, 1326
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| SpeI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Mixed up minipreps MV1 and DPRE (3 is MV1 and 4 is DPRE KAH014)
3. MV1 (E/S) = 4742, 686
4. DPRE (E/P) = 3200, 1740 (forgot that I had done s.d. mutagenesis on DPRE to make KAH014, 4/02/10)
Assemblies
- KAH222/pSB31: (1) KAH222/(E)/2614 + (9) pSB31/(E dp)/5127
- KAH223/pSB31: (2) KAH223/(E)/1537 + "
- KAH224/pSB31: (3) KAH224/(E)/1837 + "
- KAH232/pSB31: (4) KAH232/(E)/1780 + "
- KAH219/pSB31: (5) KAH219/(E)/2422 + "
- KAH220/pSB31: (6) KAH220/(E)/1345 + "
- KAH221/pSB31: (7) KAH221/(E)/1645 + "
- KAH231/pSB31: (8) KAH231/(E)/1588 + "
> PCR
--> Templates: KAH222, 223, 224, 232, 219, 220, 221, 231
--> Primers "Eco insert F1/ r1" match BB prefix/ suffix and add EcoRI to the suffix end
| Reagent |
Volume
|
| DNA (plasmid) |
1.0
|
| 10 μM primers |
1.0
|
| 2x GoTaq |
25.0
|
| dH2O |
23.0
|
--> BioRad DNA Engine, block A
- 95°C/ 3 min.
- [95°C/ 30 sec; 57°C/ 30 sec; 72 °C/ 2 min.] x30
- 72 °C/ 3 min.
- 4 °C/ ∞
> Zymo clean PCR; elute w/ 25 μL dH2O
> Digests (Fermentas FD)
--> Specific notes
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
up to 25 μL
|
| 10x buffer |
3.0
|
| EcoRI |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH222 (E PCR) |
0.728 |
1.97 |
36.4
|
| 2. KAH223 (E ") |
1.064 |
1.90 |
53.2
|
| 3. KAH224 (E ") |
0.944 |
1.81 |
47.2
|
| 4. KAH232 (E ") |
0.862 |
1.92 |
43.1
|
| 5. KAH219 (E ") |
0.803 |
1.78 |
40.2
|
| 6. KAH220 (E ") |
1.182 |
1.87 |
59.1
|
| 7. KAH221 (E ") |
0.743 |
1.79 |
37.2
|
| 8. KAH231 (E ") |
0.964 |
1.92 |
48.2
|
| 9. pSB31 (E ") |
0.991 |
1.89 |
49.5
|
> Dephosphorylation (Roche)
--> pSB31 (E)
| Reagent |
Volume
|
| DNA (clean digest) |
up to 17 μL (500 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
---
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 08/08/11
|
| 1. KAH222(E PCR)/2614, 20 ng + pSB31(E dp)/5127, 20 ng |
KAH222/pSB31 >10:1 (Pick 3)
|
| 2. KAH223(E PCR)/1537, 12 ng + " |
KAH223/pSB31 >10:1 (Pick 3)
|
| 3. KAH224(E PCR)/1837, 12 ng + " |
KAH224/pSB31 >10:1 (Pick 3)
|
| 4. KAH232(E PCR)/1780, 14 ng + " |
KAH232/pSB31 >10:1 (Pick 3)
|
| 5. KAH219(E PCR)/2422, 19 ng + " |
KAH219/pSB31 >10:1 (Pick 3)
|
| 6. KAH220(E PCR)/1345, 11 ng + " |
KAH220/pSB31 >10:1 (Pick 3)
|
| 7. KAH221(E PCR)/1645, 13 ng + " |
KAH221/pSB31 >10:1 (Pick 3)
|
| 8. KAH231(E PCR)/1588, 12 ng + " |
KAH231/pSB31 >10:1 (Pick 3)
|
| 2. pSB31(E dp)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
|
| Insert DNA |
0.6 |
0.3 |
0.3 |
0.3 |
0.5 |
0.3 |
0.4 |
0.3 |
---
|
| Vector DNA |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
|
| dH2O |
2.6 |
2.9 |
2.9 |
2.9 |
2.7 |
2.9 |
2.8 |
2.9 |
3.2 |
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp
|
Project name
Main project page
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