Matt Gethers/CRI, Thailand/Labwork/PCRs/Amplification of HmgR ORF using HotStar
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HotStar PCR
Rxn Mix
| Reagent | Volume/Amount |
| Paeru Genomic DNA Template | 0.5 μl |
| HotStar Polymerase | 1.0 μl |
| BT2736 | 1 μl 50 μM Stock |
| BT2737 | 1 μl 50 μM Stock |
| 5X Buffer | 10 μl |
| Mg2+ | None for now, may optimize later |
| 5X Q-Solution | 10 μl |
| dH20 | 26.5 μl |
| Total | 50 μl |
Rxn Conditions
Annealing Temperature: 50oC (5 degrees below 2736 annealing temp)
Extension Time: 1 minute (1 min/kb, 804 bases)
Cycle
| Step | Temperature | Duration | Notes |
| Initial Denaturation | 95oC | 5 minutes | |
| Repeat Cyclic Steps 35x | |||
| Cyclic Denaturation | 94oC | 15 Seconds | |
| Cyclic Annealing | 50oC | 1 minute | |
| Cyclic Extension | 72oC | 1 minute | |
| Repeat Cyclic Steps 35x | |||
| Final Extension | 72oC | 10 minutes |
Run Notes
7.23.08
I ran two PCRs to amplify the HmgR ORF: one with Buffer Q and the other without. I made a master mix, then aliquotted 40 μl to each of two tubes. I then added 10 μl Buffer Q to one and the same volume of water to the other. Used the reaction conditions as written. Gel results here.
8.2.08
I ran one PCR to amplify the HmgR ORF with buffer Q. I just made the reaction as in the protocol and ran with the conditions written above.
