8/26/10
9:00 AM
Dilutions to 20 ng/ul
| Sample |
concentration |
Amount sample |
Amount water |
| pArsRF1R2SDM2 1 |
339.3 ng/μL |
1.18 μL |
18.82 μL |
| pArsRF1R2SDM2 2 |
344.2 ng/μL |
1.16 μL |
18.84 μL |
| pRpoS438SDM2 2 |
362.9 ng/μL |
1.10 μL |
18.90 μL |
9:35 AM
Verification PCR
| Ingredient |
Amount |
Mastermix (5 reactions) |
| 10X buffer |
2 μL |
10 μL |
| 10 mM dNTP |
.4 μL |
2 μL |
| Template |
1 μL (20 ng) |
added individually to each reaction |
| F primer |
.4 μL |
2 μL |
| R Primer |
.4 μL |
2 μL |
| Polymerase |
.3 μL |
1.5 μL |
| H2O |
15.5 μL |
77.5 μL |
Per Reaction: 19 μL mastermix, 1 μL template
Samples:
- pArsRF1R2SDM2 1
- pArsRF1R2SDM2 2
- pRpoS438SDM2 2
- negative control (no template)
Primers:
VF2, VR at 10 micromolar
Program:
| 95°C |
3 min |
| 95°C |
30 sec |
| 55°C |
30 sec |
| 72°C |
45 sec |
| go to step 2 |
30X |
| 72°C |
2 min |
| hold at 4°C |
12:50 PM
Digestion
B0015:
| DNA |
1.7 μL (~340ng) |
| H20 |
40.8 μL |
| Buffer4 |
5 μL |
| BSA |
.5 μL |
| EcoRI |
1 μL |
| SpeI |
1 μL |
ArsRSDM2
| DNA |
17 μL (~340ng) |
| H20 |
25.5 μL |
| Buffer4 |
5 μL |
| BSA |
.5 μL |
| XbaI |
1 μL |
| PstI |
1 μL |
pSB1T3
| DNA |
.71 μL (~340 ng) |
| H20 |
41.79 μL |
| Buffer4 |
5 μL |
| BSA |
.5 μL |
| EcoRI |
1 μL |
| SpeI |
1 μL |
Incubate at 37°C for 15 min, inactivate at 80°C for 20 min.
1:00 PM
PCR ArsB
| Ingerdient |
Amount |
Mastermix (6 reactions) |
| 10X Buffer |
2 μL |
12 μL |
| 10 mM dNTPs |
.4 μL |
2.4 μL |
| Template |
.9 μL (18 ng) |
added individually to each reaction |
| F Primer |
.4 μL |
2.4 μL |
| R Primer |
.4 μL |
2.4 μL |
| Polymerase |
.3 μL |
1.8 μL |
| H2O |
14.6 μL |
87.6 μL |
| DMSO |
1 μL |
6 μL |
Samples:
- ArsB 1
- ArsB 2
- ArsB 3
- ArsB 4
- negative control (no template)
Primers:
ArsB F 2.0, ArsB R 2.0 at 10 micromolar
Program:
| 95°C |
3 min |
| 95°C |
30 sec |
| 53°C |
30 sec |
| 72°C |
2 min |
| go to step 2 |
32X |
| 72°C |
5 min |
| hold at 4°C |
2:00 PM
Gel
| Lane |
Sample |
Amount |
| 1 |
1 Kb ladder |
1 μL |
| 2 |
pArsRF1R2SDM2 1 |
5 μL sample, 1 μL dye |
| 3 |
pArsRF1R2SDM2 2 |
5 μL sample, 1 μL dye |
| 4 |
pRpoS438SDM2 2 |
5 μL sample, 1 μL dye |
| 5 |
negative control |
5 μL sample, 1 μL dye |
Ligation
| ArsRSDM2 |
2 μL |
| B0015 |
2 μL |
| pSB1T3 |
2 μL |
| H2O |
11 μL |
| Ligation Buffer |
2 μL |
| Ligase |
1 μL |
| |
|
incubate at room temperature for 10 min, inactivate at 80°C for 20 min.
2:35 PM
Transformation
50 μL cells, 2 μL ligation product. 10 min on ice, 45 sec at 42°C, 2 min on ice. add 1 mL LB, recover for 1 hour at 37°C shaking.
3:10 PM
PCR Cleanup Qiagen kit
pRpoS438SDM2 2: 21.6 ng/μL
pArsRF1R2SDM2 1: 21ng/μL
pArsRF1R2SDM2 2: 22.7 ng/μL
3:45 PM
Sequencing reactions
- 20-30 ng DNA
- total volume 10 μL
- 20 pmol primer
|
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