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Team:Calgary/21 July 2010
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'''Wednesday July 21, 2010''' | '''Wednesday July 21, 2010''' | ||
| - | + | <u> Raida: </u> | |
| + | |||
| + | Objective: PCR out native MAlE/ MalE 31 protein out of E.coli | ||
| + | Purpose: Testing Mal E/ Mal E31 primers to get 1188 bp long gene of interest. Primers that are used are gene specific and anneals right on the start of the Mal E gene | ||
| + | |||
| + | '''Method: Preparing the Master Mix''' | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | ! Reagent | ||
| + | ! x1μL | ||
| + | ! x8μL | ||
| + | |- | ||
| + | | 10x PCR Mg<sup>2+</sup> | ||
| + | | 5.0 | ||
| + | | 40.0 | ||
| + | |- | ||
| + | | 2 mm dNTP | ||
| + | | 5.0 | ||
| + | | 40.0 | ||
| + | |- | ||
| + | | 50 mm MgCl<sub>2</sub> | ||
| + | | 1.5 | ||
| + | | 12.0 | ||
| + | |- | ||
| + | | ddH<sub>2</sub>O | ||
| + | | 36.3 | ||
| + | | 290.4 | ||
| + | |- | ||
| + | | MalE-N-F 10 mm | ||
| + | | 1.0 | ||
| + | | 8.0 | ||
| + | |- | ||
| + | | MalE-N-R 10 mm | ||
| + | | 1.0 | ||
| + | | 8.0 | ||
| + | |- | ||
| + | | Platinum Taq Polymerase | ||
| + | | 0.2 | ||
| + | | 1.6 | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | '''PCR Conditions''' | ||
| + | Cycle 1: 94°C 2:00 min | ||
| + | Cycle 2: | ||
| + | *Stage 1:94°C for 1:00 min | ||
| + | *Stage 2:52°C for 0:45 min | ||
| + | *Stage 3:72°C for 1:15 min | ||
| + | Cycle 3: 72°C for 10:00 min | ||
| + | Holding Temp: 4°C | ||
| + | |||
| + | |||
}} | }} | ||
Revision as of 21:46, 21 July 2010
Wednesday July 21, 2010
Raida:
Objective: PCR out native MAlE/ MalE 31 protein out of E.coli Purpose: Testing Mal E/ Mal E31 primers to get 1188 bp long gene of interest. Primers that are used are gene specific and anneals right on the start of the Mal E gene
Method: Preparing the Master Mix {
- ! Reagent ! x1μL ! x8μL
