β-Actin mRNA Quantitation
OPTIMIZATION
Purpose: To determine the ideal dilution of samples to be used for subsequent qPCR experiments using these samples. Only dilution standards will be measured today, to determine the ideal dilution that will give a sufficiently low Ct, as well as enough sample for the entire experiment. Once determined, samples will be diluted and measured for each gene.
Dilute the primers
- β-Actin 20 μM stock. Final concentration of primers in reaction is 0.8 μM. Annealing temperature is 60°C.
Standards
- Pool 3 μl from each of 42 samples. 42x3μl = 126μl
* 1 -- 126μl Pool -- (1)
* 1:2 -- 30μl of Std 1 + 30μl H2O
* 1:3 -- 30μl of 1:2 + 15μl H2O
* 1:5 -- 30μl of 1 + 120μl H2O -- (0.2)
* 1:25 -- 30μl of 1:5 + 120μl H2O -- (0.04)
* 1:125 -- 30μl of 1:25 + 120μl H2O -- (0.008)
* 1:625 -- 126μl of 1:125 + 120μl H2O -- (0.0016)
Mastermix
| mouse β-Actin | Volume (μl) | Sample # | Final Volume (μl) |
|---|---|---|---|
| iQ SYBR Green Supermix | 12.5 | 18 | 225 |
| β-Actin Forward Primer (20μM) | 1 | 18 | 18 |
| β-Actin Reverse Primer (20μM) | 1 | 18 | 18 |
| Sterile water | 9.5 | 18 | 171 |
| Final Volume (-DNA) | 24 | Final Volume | 432 |
* Assay
- Add 24μl each of Master mix to all wells.
- Add 1μl cDNA of each sample, in duplicate. Follow template:
| 1 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 1 | 1:5 | 1:25 | 1:125 | 1:625 | NTC | 2x | 3x | ||||
| B | " | " | " | " | " | " | " | " | ||||
| C | ||||||||||||
| D | ||||||||||||
| E | ||||||||||||
| F | ||||||||||||
| G | ||||||||||||
| H |
PCR Conditions
Dye: SYBR Green
1 hold -- 95°C -- 3 minutes
40 cycles -- 95°C -- 15 seconds
40 cycles -- 60°C -- 30 seconds
40 cycles -- 72°C -- 30 seconds
+ Dissociation Curve
β-Actin Quantitation
Dilute Samples
- Dilute samples 5x
- V2/V1 = DF; V2 = V1 x 5; H2O = V2 - V1;
Mastermix
| mouse β-Actin | Volume (μl) | Sample # | Final Volume (μl) |
|---|---|---|---|
| iQ SYBR Green Supermix | 12.5 | 102 | 1275 |
| β-Actin Forward Primer (20μM) | 1 | 102 | 102 |
| β-Actin Reverse Primer (20μM) | 1 | 102 | 102 |
| Sterile water | 9.5 | 102 | 969 |
| Final Volume (-DNA) | 24 | Final Volume | 2448 |
* Assay
- Add 24μl each of Master mix to all wells.
- Add 1μl cDNA of each sample, in duplicate. Follow template:
| 1 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 1 | 1:5 | 1:25 | 1:125 | 1:625 | NTC | 1 | 2 | 3 | 4 | 5 | 6 |
| B | " | " | " | " | " | " | " | " | " | " | " | " |
| C | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| D | " | " | " | " | " | " | " | " | " | " | " | " |
| E | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| F | " | " | " | " | " | " | " | " | " | " | " | " |
| G | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | 41 | 42 |
| H |
PCR Conditions
Dye: SYBR Green
1 hold -- 95°C -- 3 minutes
40 cycles -- 95°C -- 15 seconds
40 cycles -- 60°C -- 30 seconds
40 cycles -- 72°C -- 30 seconds
+ Dissociation Curve