8/10/10
9:30
PCR
Ingredient |
Amount |
Mastermix (4 reactions) |
10X Buffer |
2 μL |
8 μL |
10 mM dNTPs |
.4 μL |
1.6 μL |
Template |
.9 μL (18 ng) |
3.6 μL |
F Primer |
.4 μL |
added individually to each reaction |
R Primer |
.4 μL |
added individually to each reaction |
DMSO |
1 μL/td>
| 4 μL |
Polymerase |
.3 μL |
1.2 μL |
H2O |
14.6 μL |
58.4 μL |
Per Reaction: 19.2 μL Mastermix, .4 μL F Primer, .4 μL R Primer.
Samples:
- LamB F1R
- LamB F2R
- Negative control (no primer)
Primers:
- LamBF1Xba
- LamBF2Xba
- LamBRSpe
Program:
95°C |
3 min |
95°C |
30 sec |
53°C |
30 sec |
72°C |
2 min |
go to step 2 |
31X |
72°C |
5 min |
hold at 4°C |
10:20 AM
PCR cleanup
Qiagen Kit
Concentrations:
- pArsRF1R2 2' 28.8 ng/μL
- pARsRF1R2 1 24 ng/μL
- pArsRF1R2 2 30 ng/μL
- pRpoS438 1 8 ng/μL
- pRpoS438 2 7 ng/μL
11:00 AM
Site Directed Mutagenesis PCR
Ingredient |
Amount |
Mastermix (6 reactions) |
10X Buffer |
5 μL |
30 μL |
10 mM dNTPs |
1 μL |
6 μL |
Template |
20 ng |
added individually to each reaction |
Primer 1 |
2 μL |
added individually to each reaction |
Primer 2 |
2 μL |
added individually to each reaction |
Polymerase |
1 μL |
6 μL |
H2O |
35 μL |
210 μL |
Samples:
- pArsRF1R2 2'
- pARsRF1R2 1
- pArsRF1R2 2
- pRpoS438 1
- pRpoS438 2
Program:
95°C |
3 min |
95°C |
30 sec |
64°C |
1 min |
72°C |
1 min |
go to step 2 |
12X |
72°C |
1 min |
hold at 4°C |
Primers:
10 micromolar
- SDM2 pArsR RFC10 F502
- SDM1 pArsR RFC10 F502
- SDM2 pRpoS RFC10 F438
- SDM2 pRpoS RFC10 F438
1:35 PM
Gel
Lane |
Sample |
Amount |
1 |
1 Kb Ladder |
1 μL |
2 |
LamBF1R |
5 μL sample, 1 μL dye |
3 |
LamBF2R |
5 μL sample, 1 μL dye |
4 |
LamBF2R |
5 μL sample, 1 μL dye |
5 |
negative control |
5 μL sample, 1 μL dye |
PCR cleanup:
- pArsRF1R2 2'
- pARsRF1R2 1
- pArsRF1R2 2
- pRpoS438 1
- pRpoS438 2
all around 4 ng/μL
Dpn1 Digestion:
DNA |
28 μL |
H2O |
15.5 μL |
Buffer 4 |
5 μL |
BSA |
.5 μL |
DpnI |
1 μL |
incubate at 37°C for 1 hour, inactivate at 80°C for 20 min.
Samples:
- pArsRF1R2 2'
- pARsRF1R2 1
- pArsRF1R2 2
- pRpoS438 1
- pRpoS438 2
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