06/06/11
- ✓ Assemblies: full miR sensors w/ different promoters/ binding site copy numbers
- ✓ DH5α Turbo chemically competent cell prep: streak LB (plain) plates with left-over cells w/o DNA (from transformations)
Assemblies
- KAH186/196: KAH186/(E/S)/2025 ✓ + (1) KAH196/(E/X)/7806
- KAH184/196: KAH184/(E/S)/2191 ✓ + "
- KAH187/196: KAH187/(E/S)/2269 ✓ + "
- KAH188/196: KAH188/(E/S)/2124 ✓ + "
- KAH189/196: KAH189/(E/S)/2296 ✓ + "
- KAH190/196: KAH190/(E/S)/2368 ✓ + "
- KAH191/196: KAH191/(E/S)/2223 ✓ + "
- KAH183/196: KAH183/(E/S)/2383 ✓ + "
- KAH192/196: KAH192/(E/S)/2461 ✓ + "
- KAH186/177: KAH186/(E/S)/2025 ✓ + KAH177/(E/X)/7978 ✓
- KAH187/177: KAH187/(E/S)/2269 ✓ + "
- KAH188/177: KAH188/(E/S)/2124 ✓ + "
- KAH189/177: KAH189/(E/S)/2296 ✓ + "
- KAH190/177: KAH190/(E/S)/2368 ✓ + "
- KAH191/177: KAH191/(E/S)/2223 ✓ + "
- KAH192/177: KAH192/(E/S)/2461 ✓ + "
- KAH186/198: KAH186/(E/S)/2025 ✓ + (2) KAH198/(E/X)/8050
- KAH184/198: KAH184/(E/S)/2191 ✓ + "
- KAH187/198: KAH187/(E/S)/2269 ✓ + "
- KAH188/198: KAH188/(E/S)/2124 ✓ + "
- KAH189/198: KAH189/(E/S)/2296 ✓ + "
- KAH190/198: KAH190/(E/S)/2368 ✓ + "
- KAH191/198: KAH191/(E/S)/2223 ✓ + "
- KAH183/198: KAH183/(E/S)/2383 ✓ + "
- KAH192/198: KAH192/(E/S)/2461 ✓ + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
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 (E/X) |
1.617 |
1.88 |
80.9
|
2. KAH198 (E/X) |
1.353 |
1.85 |
67.6
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 06/07/11
|
1-9. KAH###(E/S)/~2200, 11 ng + KAH196(E/X)/7806, 20 ng |
Notes: >10:1 for all plates
|
10. KAH196(E/X)/ 25 ng |
|
11-17. KAH###(E/S)/~2200, 11 ng + KAH177(E/X)/7978, 20 ng |
Notes: >10:1 for all plates
|
18. KAH177(E/X)/ 25 ng |
|
19-27. KAH###(E/S)/~2200, 11 ng + KAH198(E/X)/8050, 20 ng |
Notes: >10:1 for all plates
|
28. KAH198(E/X)/ 25 ng |
|
|
1-9 |
10 |
11-17 |
18 |
19-27 |
28
|
Insert DNA |
3.0 |
--- |
3.0 |
--- |
3.0 |
---
|
Vector DNA |
0.25 |
0.25 |
1.0 |
1.0 |
0.3 |
0.3
|
2x lgn buf (Roche) |
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 |
|
dH2O |
0.75 |
3.75 |
--- |
3.0 |
0.7 |
3.7
|
|
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> Make master mix w/ lign. buffer, vector DNA; add insert DNA to individual tubes; add ligase to master mix and quickly aliquot
--> Add rxn. to 25 μL DH5α Turbo cells
|