Is the region you are trying to amplify GC rich ? Some polymerases come with regular and GC compatible buffers for this issue. Since you designed the primers using the tool I'll assume the primer pair is around 25bp and Tm within 5oC of each other and is cleared for self-complementarity and hairpins. If you are not getting any amplicons you can check the pair using hot-start polymerases or do a touch down PCR. If you still dont get any amplicons, then the batch you recieved is most likely bad.

A couple of times I recieved faulty batches of primers that didnt produce any amplicons. Once I got it replaced they worked fine. I am including a checklist that I use for primer design. You can follow along and see if there is a step you missed while designing. If all the steps are fine then the problem could be with the batch you recieved.

1. Designing primers for expressing a particular sequence. Expression of any given protein within a bacterial system depends on its size, hydrophobicity, number and position of repeat sequences. These parameters need to be kept in mind while choosing the sequence of interest. You should check the secondary structure before designing the forward primer. (http://www.biogem.org/tool/chou-fasman/) . Starting your protein from a beta sheet is most preferred. It is best not to initiate from helix, random coils or turns. This affects the rate of bacterial translation and eventually your yield.

2. Choose the restriction enzymes needed. Keep in mind to check whether the restriction enzyme that flanks the initial residues of your gene is in frame with the vector’s start site. If not, then add extra nucleotides after your restriction site to bring the gene within the reading frame.

3. Addition of restriction enzyme flanking sequences. Choose your flanking sequences from this list - (http://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf). Two things need to be noted here. First, try to incorporate large flanking sequences. You can design a primer that has a Tm upto 60C. Therefore, you can afford to keep flanking sequences of upto 3 or even 4 nucleotides. Secondly, it is seen that when you put two flanking sequences on either side of your restriction site (ie. GGAAGATCTTCCgene-here) cloning becomes easier. The mechanism as why cloning becomes easier can be debated but if you can afford to incorporate two flanking sequences while keeping your Tm within 60C, then do so.

4. Designing the Forward primer: Now choose a stretch of about 30 nucleotides that start your gene and add it to your forward restriction site. Plug the sequence in a online Tm calculator (https://tmcalculator.neb.com/#!/) and check if the Tm is within 60C. If it exceeds this temperature, keep deleting the final nucleotides till the Tm falls to this temperature. (minimum primer size is 18bp)

5. Designing the Reverse primer: Similarly choose a stretch of about 30 nucleotides that flank your sequence of interest. Now keep in mind if you need to terminate this sequence manually or using your vector. Common vectors usually have a C-terminal tag ending in a stop codon. In such cases you don’t need to retain any stop codons in your reverse primer. Once the sequence is selected generate its reverse complement (https://www.bioinformatics.org/sms/rev_comp.html) . Take the reverse complement and attach it to your reverse primer’s restriction site. Plug the primer into a Tm calculator. Now you need to bring the Tm of your primer close to 5 degrees of your Forward primer by deleting the last few nucleotides.

6. While designing make sure your primers end in a guanine or cytosine. (refer GC Clamping).

7. Now check both primers for their potential for self-complementarity and hairpin formation. (http://www.bioinformatics.org/sms2/pcr_primer_stats.html) . Hairpins in the regions containing the restriction sites can be tolerated. But severe hairpins will prevent the polymerase from amplifying your restriction site, therefore affecting your insert digestion and consequently your ligation. Avoid hairpins that include your sequence of interest. They might hinder with your primer binding to the template and give weak amplicons.

8. If you face any hindrance with the sequence due to its unavoidable self complementarity and hairpin loops, delete the nucleotides in question and BLAST the region containing your sequence of interest against the template. (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) . Keep in mind that you don’t need to BLAST the region of your primer containing the restriction sites and its flanking sequences. Your goal is to make sure that your primer can successfully bind specifically to the sequence of interest within the template and not show any homology with an off-target site.

9. If you find that self-complementarity and hairpins are inevitable and you cannot remove the sequence due to its importance; synthesize the primer and test it. Using DMSO in your PCR reaction mix can help to reduce non-specific priming and self-complementarity issues.

10. In conclusion, You need to minimize sequence hindrances while keeping your primer within 5 degrees of Tm of each other while they remain specific to your sequence of interest.

Hi Manjari

It is true that you might have received a bad set of primers and you will have to re-order the set of primers by specifically keeping in mind the GC content but firstly try to amplify using different polymerase. I too have faced such problem with one set of primer so i reordered it.