01/25/10
- ✓ Colony PCR screen: KAH 94, 95/ JDS48, 49; HS4 primers
- ✓ Assembly: KAH140, 141/ V0201 (re-do, d.p. vector)
- ✓ Cultures: KAH94/JDS48 #1 & KAH94/JDS49 #18 (2 each)
Colony PCR screen
> Use BB_HS4 f1 & BB_HS4 r1 primers
> Controls: (+) = KAH94 (0.5μL); (-) = JDS48 (0.5μL)
| Reagent |
Volume
|
Expected:
340, 686 bp
|
15 μL/lane; 1% agarose
|
| E. coli from streak |
0.0
|
| 10μM primer mix |
1.0
|
| 2x GoTaq Green |
10.0
|
| dH2O |
9.0
|
| |
20 μL
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|
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--> BioRad 96-well:
- 95°C/ 3 min.
- [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x30
- 72°C/ 3 min.
- 4°C/ ∞
--> Check minipreps of KAH94/JDS48 #1 & KAH94/JDS49 #18 tomorrow with BglII and PstI digests
Assemblies
- KAH140/V0201: KAH140/(N/S)/2945 + V0201/(N/S, d.p.)/5157
- KAH141/V0201: KAH141/(N/S)/2939 + "
> Dephosphorylation (Roche)
--> d.p. treat V0201(N/S)
| Reagent |
Volume
|
| DNA (clean digest) |
17.0 (~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) 01/26/10
|
| 1. KAH140(N/S)/2939, 29 ng + V0201(N/S)/5127, 25 ng |
KAH140/V0201 >10:1 (Pick 2)
|
| 2. KAH141(N/S)/2527, 25 ng + " |
KAH141/V0201 >10:1 (Pick 2)
|
| 3. V0201(N/S)/ 25 ng |
|
| |
1 |
2 |
3
|
| Insert DNA |
3.0 |
3.0 |
---
|
| Vector DNA |
1.0 |
1.0 |
1.0
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0
|
| dH2O |
--- |
--- |
3.0
|
| |
10μL |
10μL |
10μL --> R.T./ 10 min.; Add 20 μL Z-DH5α
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Project name
Main project page
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