IVT pFC8, pFC14, pFC17 with new reagents: attempt 2 #
Protocol #
DNA concentrations #
Remeasured all DNA concentrations before starting today.
| Plasmid | DNA concentration (ng/ul) |
|---|---|
| pFC8 | 277 |
| pFC14 | 1356 |
| pFC17 | 1236 |
Reagents #
| Reagent | pFC8 | pFC14 | pFC17 |
|---|---|---|---|
| 10x Buffer | 2 | 2 | 2 |
| 100 mM DTT | 1 | 1 | 1 |
| 25 mM rNTP | 0.4 | 0.4 | 0.4 |
| H20 | 14.4 | 16.15 | 16.11 |
| DNA | 2.16 | 0.44 | 0.48 |
Since using more plasmids this time made a master mix composed of the constant reagents, made 1.5x more than needed. Composition is shown in the table below.
| Reagent | Volume (ul) |
|---|---|
| 10x buffer | 9 |
| DTT | 4.5 |
| rNTP | 1.8 |
Added 3.4 ul MM to each sample plus H20 and DNA specific to each plasmid.
All plasmids use the T3 polymerase.
Results #
Ran 0.8% agarose gel for 1 hour 45 mins.
