"
Illinois/1 August 2008
From 2008.igem.org
Receptor Tyrosine Kinase Group
PCR troubleshooting of yesterday's reaction: use gradient for annealing temp (40-65 degrees), use more template, leave out end primers
Four series of reactions using a gradient annealing temp were run in addition to a fifth series omitting the end primers.
| reaction series | A | B | C | D | E |
| template (5+7) from 7-30 PCR | 0.5ul | 1ul | 1.5ul | 2ul | 1ul |
| primers | 1ul of forward and reverse primers (omitted in E series) | ||||
| master mix | 20ul | ||||
| H20 | to 50ul total volume | ||||
annealing temps: 40, 43.8, 50, 54, 60, and 64 degrees
PCR program: 1. 94 degrees 5 min 2. 94 degrees 1 min 3. annealing step 1 min 4. 72 degrees 1 min 5. GOTO 2. 29 cycles 6. HOLD at 4 degrees
PCR products run on 1.5% agarose gel at 200V for ~20 minutes, then 240V for ~20 minutes.
Products in the 700-800bp range in all lanes. Perhaps it worked because of using a differnt product from the 7-30 PCR.
