Hi Bankim,

Typically you only need to add the extra bases to the 5' end of your primer.  The 3' end of your restriction site will have the entire PCR product and will not need extra bases to get efficient cutting.  You may need to add bases after the restriction site (3' to the restriction site) in order to ensure your PCR product is in the correct reading frame (if you're cloning an mRNA for protein expression).  If so, make sure you aren't accidentally adding unwanted codons (stops, starts, or unusual amino acids like proline) or other restriction sites. 

Amy

Hi Bankim,

There are many rules to design primers, mostly rely on certain parametesrs such as Tm values(should be less than 60 degree celsius for both primers), GC%(should be closer to 50%), should not form secodary structures or self annealing during PCR reactions. You can use certain online softwares such as Oligocalc or Primer3  to calculate the efficiency of your primers. You also need to add overhang regions in your primers before restriction sites(such as CTCTCT) as they allow the restriction enzymes to sit over them before initiating the digestion reaction. Without such overhangs, restriction specificity gets hampered or stop. Number of overhang nucleotides varies for certain enzymes but it remains 6 nucleotides for most of the available enzymes.

Hope it will help.

For just routine cloning experiments, you don't need primers with RE sites if  you are using commercial TA cloning kits. For protein expression studies you need primers with RE site. the common practice is to add 5-6 bases at the 5' end of the primer just before the RE site. as Amy said, you can add bases at the 3' prime end of RE sie if you need to put your CDS into correct reading frame.

Dear all,

Thank you very much for your helping answers.

But my question was, may I add any set of 6 bases of my wish at 5' end of Restriction site? This neb site (https://www.neb.com/~/media/NebUs/Files/Chart%20image/

cleavage_olignucleotides_old.pdf) is saying that only some sets of 3-6 bases at 5' end will enhance restriction digestion, not the all possible sets. Please see the case of restriction enzyme PvuI, SpeI etc of the chart can be seen fron attaches web-link.

Sincerely,

Bankim Mondal

yes you can add.

 Dear Gaurav,

Thank you very much.

I have six A in the upstream of my primer before restriction site as a reader sequence. Is that the reason I am getting trouble in amplifying my PCR product?

Why an extra nucleotide is added at 3 prime end of primer in AFLPs??