07/27/10
- ✓ Assembly: start re-cloning pAct5C/microMCS (from 7/25/10)
- ✓ Cultures/ minipreps: KAH015 (4, 2 mL)
Digests
> Mut mCh clones from last week
> Check with X/P digests (unique XbaI introduced by microMCS insert)
> Will need to confirm proper orientation of microMCS by sequencing
| Reagent |
Volume
|
Expected:
1-4.KAH015 = 649, 7753
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| BamHI |
1.0
|
| KpnI |
1.0
|
| PstI |
1.0
|
| dH2O |
8.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Send all 4 to Genewiz for sequencing on Wednesday
Assembly
--> re-do pAct5C/microMCS (from 7/25/10)
- KFV1: (1) pAct5C/(S/N)/~7000 + microMCS/(ΔN/ΔS dsOligo)/~35
> Dephosphorylation (Roche)
--> pAct5C/(S/N)
| Reagent |
Volume
|
| DNA (clean digest) |
15.0 (120 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
2.0
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 6 ng/μL
|
>Kinase rxn. and Oligo annealing
--> Need to phosphorylate microMCS before inserting into dp vector (overnight)
| Reagent |
Volume
|
| 100 μM oligos |
(3.0 μL each, up to 21 μL)
|
| 10x PNK buffer (NEB) |
3.0
|
| 10 mM ATP |
2.0
|
| T4 PNK |
2.0
|
| dH2O |
2.0
|
| |
30 μL --> 37°C/ 1 hr.; Add 4.0 μL 500 mM NaCl; 100 °C 10 min.; cool to R.T. o/n
|
7/28/10
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/27/10
|
| 1. microMCS(ΔS/ΔN dsOligo)/35, 0.25 ng + pAct5C(S/N)/7000, 25 ng |
KFV1 2 colonies
|
| 2. pAct5C(S/N), 25 ng |
0 colonies
|
| |
1 |
2
|
| Insert DNA |
0.5 |
---
|
| Vector DNA |
4.5 |
4.5
|
| 2x lgn buf (Roche) |
6.0 |
6.0
|
| T4 ligase (NEB) |
1.0 |
1.0
|
| dH2O |
--- |
0.5
|
| |
12μL |
12μL
|
|
Project name
Main project page
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