07/20/11
- ✓ Minipreps 1: KAH219, 223, 224/pSB31 (8 each)
- ✓ Cultures/ minipreps 2: KAH213, 214, 215, 219, 220, 221, 222, 223, 224/MV1 (2 each)
- ✓ Minipreps 3: High yield mp's for KAH220, 221, 222/pSB31 for Manching
- ✓ Assemblies: miR-luc sensor "196/MV8 set"
- ✓ Assemblies: Re-do KAH219, 223, 224/pSB31
Minipreps 1
> Check with KpnI/NcoI digests
| Reagent |
Volume
|
Expected:
pSB31 = 4500, 450
*pSB31 bands
**last frag + 300
1-8. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532**
9-16. 223/pSB31 5-8 = 4500*, 150*, 636, 1190**
17-24. 224/pSB31 5-8 = 4500*, 150*, 636, 1490**
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> None worked. Re-do ligations.
Minipreps 2
> Check with E/P digests
> See 2/28/22 for example
| Reagent |
Volume
|
Expected:
Vector frag. = 4072
Insert + 1326...
1,2. KAH213/MV1 = 4160
3,4. KAH214/MV1 = 3083
5,6. KAH215/MV1 = 3383
7,8. KAH219/MV1 = 3748
9,10. KAH220/MV1 = 2671
11,12. KAH221/MV1 = 2971
13,14. KAH222/MV2 = 3940
15,16. KAH223/MV1 = 2863
17,18. KAH224/MV1 = 3163
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Pick more colonies for 213, 219/MV1 (3 each)
Minipreps 3
> From 5 mL cultures from spot-streaks; combine duplicates
> Check with KpnI/NcoI digests
| Reagent |
Volume
|
Expected:
1. KAH220/pSB31 = 4500, 150, 444, 1190
2. KAH221/pSB31 = 4500, 150, 444, 1490
3. KAH222/pSB31 = 4500, 150, 636, 1735
See 7/18/11
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH220/pSB31 mp |
2.229 |
1.86 |
111.4
|
| 2. KAH221/pSB31 mp |
0.893 |
1.92 |
44.7
|
| 3. KAH222/pSB31 mp |
0.893 |
1.92 |
44.7
|
--> Target concentration is 500 ng/μL. Concentrations too low. Zymo clean & concentrate tomorrow. Re-do digest.
Assemblies
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> Re-do digest of 196/MV8 and 198/MV8
--> Today, do 196/MV8 ligations
- KAH186-196/MV8: KAH186/(E/S)/2025 ✓ + (1) KAH196/MV8(E/X)/7978
- KAH184-196/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH187-196/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH188-196/MV8: KAH188/(E/S)/2124 ✓ + "
- KAH189-196/MV8: KAH189/(E/S)/2296 ✓ + "
- KAH190-196/MV8: KAH190/(E/S)/2368 ✓ + "
- KAH191-196/MV8: KAH191/(E/S)/2223 ✓ + "
- KAH183-196/MV8: KAH191/(E/S)/2223 ✓ + "
- KAH192-196/MV8: KAH192/(E/S)/2461 ✓ + "
(2) KAH198/MV8(E/X)/8050 (vector for next set)
> Digests (Fermentas FD)
| Reagent |
Volume |
|
|
| DNA (plasmid) |
up to 25 μL
|
| 10x buffer |
3.0
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH196/MV8 (E/X) |
0.711 |
1.87 |
35.6
|
| 2. KAH198/MV8 (E/X) |
0.893 |
1.92 |
44.7
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/14/11
|
| 1-9. KAH###(E/S)/~2200, 11 ng + KAH196/MV8(E/X)/7978, 20 ng |
Notes: #:1 for all plates
|
| 10. KAH196/MV8(E/X)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
| Insert DNA |
1.0 |
1.0 |
1.5 |
1.0 |
1.0 |
0.5 |
1.0 |
0.5 |
0.8 |
---
|
| Vector DNA |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
0.6
|
| 2x lgn buf (Roche) |
5.0 |
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 |
1.0
|
| dH2O |
2.4 |
2.4 |
1.9 |
2.4 |
2.4 |
2.9 |
2.4 |
2.9 |
2.6 |
3.4
|
| |
10 μL |
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
Assemblies
> Repeat from 7/17/11
> Use max vol. insert for 12 μL rxns.
- KAH219/pSB31: KAH219/(E/S blunt)/2422 ✓ + pSB31/(E blunt dp)/5127 ✓
- KAH223/pSB31: KAH223/(E/S blunt)/1537 ✓ + "
- KAH224/pSB31: KAH224/(E/S blunt)/1837 ✓ + "
> Ligations
| Ligation |
Plate results (lig : neg crtl) 01/23/11
|
| 1. KAH219(E/S blunt)/2422, 24 ng + pSB31(E blunt dp)/5127, 25 ng |
KAH219/pSB31 2:1 (Pick 4)
|
| 5. KAH223(E/S blunt)/1537, 15 ng + " |
KAH223/pSB31 2:1 (Pick 4)
|
| 6. KAH224(E/S blunt)/1837, 18 ng + " |
KAH224/pSB31 5:1 (Pick 4)
|
| 7. pSB31(E blunt dp)/ 25 ng |
|
| |
1 |
2 |
3 |
4
|
| Insert DNA |
4.0 |
4.0 |
4.0 |
---
|
| Vector DNA |
1.0 |
1.0 |
1.0 |
1.0
|
| 2x lgn buf (Roche) |
6.0 |
6.0 |
6.0 |
6.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
--- |
--- |
--- |
4.0
|
| |
10 μL |
10 μL |
10 μL |
10 μL
|
--> ~1 hr/ r.t.; Add to 30 μL DH5α turbo
|
Project name
Main project page
Previous entry Next entry
