Are the restriction sites already in your plasmid and you just need to add them to your gene? If so, you would just design them into the 5' ends of your primers, adding several additional random bases upstream for the enzyme to have something to grab onto. Pay attention to the orientation in your forward/reverse primers and test it in a software like SnapGene before you order them.

If you want an easier cloning method, look into Gibson cloning. It uses primers for both the vector and insert with overlapping sequence, eliminating the need for restriction sites all together and allowing you to insert your gene almost anywhere. It also has a much higher efficiency than restriction cloning. An example is the HiFi assembly kit by NEB.

Hello,

The pGEM-T Easy plasmid contains a good polylinker with sufficient possibility of choosing cloning sites.

Therefore, you must add the sequences of the restriction sites that you have chosen to carry out your cloning, when designing your primers.

Also add a few bases upstream of the restriction sites that you have added to allow your enzymes to function properly.

Thank you for your reply

Yes restriction sites are present in both of the plasmids.

Another doubt that had should I add few random bases between the two restriction enzyme that i am going to attach to primers. As both the restriction enzyme are for two different vectors

Also, as I am going to attached two restriction enzyme to my primers having more GC bases . Can the GC percentage of a primer be more than 60?

60 for a single primer? good primers have a size of 18 to 23 nucleotides after which there are the bases of your sites which contain 6 or 8 bases.

For GCs it will mainly influence the Tm that you will use to calculate the hybridization temperature for your PCR. If the GC content is large, so will the hybridization temperature.

However, you must try to balance the GC content of the 2 primers so that their Tm (and therefore their hyridation temperatures) are close.