how to add type II restriction site sequence in primer for PCR amplification? how much size can be add-in a plasmid?
To add a Type II restriction site sequence in a primer for PCR amplification, you need to design the primer with the desired restriction site sequence at the 5' end or 3' end. The restriction site sequence should be added in such a way that it does not interfere with the binding of the primer to the template DNA.
Here is an example of how you can add a Type II restriction site sequence to a primer:
Suppose you want to add a BamHI restriction site (GGATCC) to the 5' end of your primer. You can design your primer with the following sequence:
5'-GGATCCNNNNNN-3'
Here, the "NNNNNN" represents the sequence of your specific primer. The BamHI restriction site is added at the 5' end of the primer, which will allow the PCR product to be digested with BamHI.
As for the size that can be added in a plasmid, it depends on the size of the plasmid and the specific restriction enzyme site you want to add. Generally, plasmids can accommodate several kilobases of additional DNA, but the size limit can vary depending on the plasmid and the desired application. It's important to consider the size of the insert and the size of the plasmid to ensure that the plasmid remains stable and that the insert does not interfere with essential plasmid functions.
In addition to what Ali Raza says it is a good idea to add 3 random bases to the 5' end of each primer(including the cut site) so that the enzyme binds and cuts better so the primer sequence is 3'primer-cutsite-nnn and do not forget that the annealing temperature of the primer should not include the cut site or Ns
There is not really any size limit for a plasmid, but it just gets harder to manipulate and transform as it gets larger. Cosmids are nearly 50kb in size and build using standard E. coli plasmids, for example.
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