Gaurav Agrahari ,

How did you design the first primer set? Are these to be used in Real Time PCR, Sequencing, just normal PCR, etc?

When designing Primers, I dont use online tools besides designing primers with certain rules that I follow:

1) Primer length 18-25 bp

2) Annealing temperature close to 60 C

3) GC content under 50%

4) No self complimentary regions

5) No predicted Hairpin

6) If used to RT-qPCR analysis, I design primers to fall within Exon-exon junctions and have total amplicon around 180 bp

For designing Primers I use:

ApE (A Plasmid Editor Software) to visually design primers

Check Primer specifications with OligoCalc http://biotools.nubic.northwestern.edu/OligoCalc.html

And check specificity using the UCSC Genome Browser In-silico PCR

https://genome.ucsc.edu/cgi-bin/hgPcr

Otherwise if you dont want to manually build the ideal primer, you can use websites like the following:

http://bioinfo.ut.ee/primer3-0.4.0/

https://www.idtdna.com/pages (Primer Design Tool)

Following