20.109(S08):Site-directed mutagenesis (Day2)
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.
- Read through the following protocol and prepare all calculations before beginning physical manipulations of your samples.
- You will be given an X μL mixture that contains buffer and dNTPs when you are ready to begin. Aliquot 40 μL per tube.
- Add 2 μL of template DNA (“IPC plasmid”) to each of your three reaction tubes. Do not add template to your control tubes!
- Note: mutagenesis reactions are expected to run smoothly with 5-50 ng of plasmid DNA. You have been given a 1:200 dilution of miniprep DNA.
- Increase the volume of each reaction to 50 μL by adding the appropriate amount of water (note: this amount is different for sample versus control reactions).
- Finally, add 1 μL of PfuTurbo DNA polymerase to each reaction.
- Once each group is ready, we will begin the thermocycler, under the following conditions:
Segment | Cycles | Temperature (° C) | Time |
---|---|---|---|
1 | 1 | 95 | 30 sec |
2 | 18 | 95 | 30 sec |