Set up PCR for single/multigene

Purpose

Perform Polymerase Chain Reaction to amplify a fragment of a targeted gene

Prepare the working solution of your target primers.
Stock primers are ordered at a concentration of 100mMol and 25ng.
General practice is to prepare 5mMol primers pair from the stock solution. (Add 5ul of Forward and 5ul of Reverse primers to 90ul of Nucleus Free water to obtain a final 5mMol concentration)
Following are the general composition and constituents to set up a PCR reaction.

NOTE: For Sanger’s sequencing, Medauxin and Aggrigenome asks for a total of 20ul final reaction volume; modify accordingly.

Make sure to UV the pipettes, tips and strip tubes before you start setting up the PCR.
We have two PCR machines (Eppendorf) in the lab, use one of them to run your reactions.
While setting up for the first time; create a new user with your name and save the desired programme.
Book the machine slots by using the QR code available next to PCR machine.
Typical programme runs for about 2 to 2.5 hours, but varies depends on the number of cycles.
Once the programme finishes running, visualize the PCR product by running on 1.5% agarose gel to confirm the amplicon size and success of PCR. (Refer next section “Gel” for further details)
Clean the pipettes and your work bench after you finished working.