01/24/10
- ✓ Minipreps: KAH140, 141/V0201 (4 each)
- ✓ PCR colony screen: KAH94, 95/ JDS48, 49 plates
Minipreps
> Check with E/P digests (Oops, cut with E/P instead of XbaI)
--> E/P cuts 94 bp fragment from backbone vector
| Reagent |
Volume
|
Expected:
Empty vector = ~5000
1-4. KAH140/V0201 = ~7945
5-8. KAH141/V0201 = ~7939
|
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.
|
PCR colony screen
> Use RFP1 primer mix
> Controls: (+) = KAH94 (0.5 μL); (-) = JDS48 (0.5 μL)
| Reagent |
Volume
|
Expected:
100 bp
|
15 μL/lane; 1% agarose
|
| Colony pick |
0.0
|
| 10 μM primer mix |
1.0
|
| 2x GoTaq Green |
10.0
|
| dH2O |
9.0
|
| |
20 μL
|
| |
|
| |
|
| |
|
--> BioRad 96-well: 95°C/ 3 min.; [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x30; 72°C/ 3 min.
--> All look positive. Tomorrow check streaks for HS4
|
Project name
Main project page
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