Difference between revisions of "20.109(S08):Site-directed mutagenesis (Day2)"
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#Calculate the dilution of primer that you need to prepare in order to have 125 ng present in 2.5 μl. Prepare 100-200 μL of this dilution and keep it on ice. | #Calculate the dilution of primer that you need to prepare in order to have 125 ng present in 2.5 μl. Prepare 100-200 μL of this dilution and keep it on ice. | ||
| − | ===Part 2: site-directed mutagenesis== | + | ===Part 2: site-directed mutagenesis=== |
We will be using the QuickChange® kit from Stratagene to perform our site-directed mutageneses. Each group will set up three reactions and associated controls. You should work quickly but carefully, and keep your tubes in a chilled container at all times. | We will be using the QuickChange® kit from Stratagene to perform our site-directed mutageneses. Each group will set up three reactions and associated controls. You should work quickly but carefully, and keep your tubes in a chilled container at all times. | ||
==For next time== | ==For next time== | ||
Revision as of 19:53, 13 December 2007
Contents
Introduction
Protocols
Part 1: primer preparation
- Calculate the amount of water needed for each primer to give a concentration of 1 ng/mL.
- Touch-spin your primers, resuspend each in the appropriate volume of water, and touch-spin again.
- Calculate the dilution of primer that you need to prepare in order to have 125 ng present in 2.5 μl. Prepare 100-200 μL of this dilution and keep it on ice.
Part 2: site-directed mutagenesis
We will be using the QuickChange® kit from Stratagene to perform our site-directed mutageneses. Each group will set up three reactions and associated controls. You should work quickly but carefully, and keep your tubes in a chilled container at all times.
