03/17/14
- Luc 14 genomic DNA test - restriction digest and gel
- Luc 14 genomic DNA clean-up & PCR
Luc 14 genomic DNA test - restriction digest and gel
- Check genomic DNA on a gel, with or without restriction digestion
- Luc 14 HEK - No enzyme
- Luc 14 HEK - EcoRI (fast digest)
- Luc 14 HEK - PstI (fast digest)
- U2OS plain - No enzyme
- U2OS plain - EcoRI (fast digest)
- U2OS plain - PstI (fast digest)
| REAGENT |
1 |
2 |
3 |
4 |
5 |
6
|
Inconclusive: Gel showed no DNA smear
|
| Genomic DNA (~500 ng) |
2.0 μL |
2.0 |
2.0 |
1.0 |
1.0 |
1.0
|
| Rest. enzyme |
0 |
2.0 |
2.0 |
0 |
2.0 |
2.0
|
| 10x buffer |
2.0 |
2.0 |
2.0 |
2.0 |
2.0 |
2.0
|
| dH2O |
16.0 |
14.0 |
14.0 |
17.0 |
15.0 |
15.0
|
| |
20.0 |
20.0 |
20.0 |
20.0 |
20.0 |
20.0
|
Luc 14 genomic DNA clean-up & PCR
- Clean up 1/2 of the Luc genomic DNA (50 μL) on a Zymo clean and concentrator column
- 50 μL ~ 4340 ng
- Eluted with 20 μL dH2O ~ 217 ng/μL
- GAPDH A3 - #359, 360
- GAPDH B2 - #361, 362
- MMP12 A3 - #325, 326
- MMP12 B2 - #367, 368
- MMP12 C2 - #327, 328
- luc 1 - luc f1, luc r1 - #724, 725
- luc 2 - luc f2, luc r2 - #726, 727
- luc 4 - luc f4, luc r4 - #730, 731
- Kozak plasmid + P0001/P0002
- Kozak plasmid + P0001/P0002 (again)
| Reagent |
Kozak |
Luc14
|
EXPECTED
1. GAPDH A3 = 125 bp
2. GAPDH B2 = 112
3. MMP12 A3 = 100
4. MMP12 B2 = 118
5. MMP12 C2 = 103
6. luc 1 = 110
7. luc 2 = 98
8. luc 4 = 105
9, 10. Kozak = 277
|
2% agarose gel, 20 μL/lane, 1kb+ Ladder
|
| DNA |
0.5 μL |
0.5
|
| P0001 |
1.0 |
---
|
| P0002 |
1.0 |
---
|
| mixed primers |
--- |
1.0
|
| 2x GoTaq |
12.5 |
12.5
|
| dH2O |
10.0 |
11.0
|
| |
25.0 |
25.0
|
PCR
- 95°C, 60 sec
- [95°C, 15 sec; 57°C, 15 sec; 72°C 15 sec] x35
- 72°C, 60 sec
- 4°C ∞
|
Project name
Main project page
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